Wythe was born in 1726 at Chesterville, the plantation operated by three generations of the Wythe family in what was then Elizabeth City County but is now Hampton, Virginia. His maternal great-grandfather was George Keith, a Quaker minister and early opponent of African slavery, who returned to the Church of England but was sent back as a missionary to the East Coast before ultimately returning to England. His mother, Margaret Walker of Kecoughtan, a learned woman probably raised as a Quaker, instilled a love of learning in her son. In his later years, Wythe became known for his outdated Quaker dress, as well as his gentle manner, which could cause even a surly dog to "unbend and wag his tail." After the early death of his father, Wythe probably attended grammar school in Williamsburg before beginning legal training in the office of his uncle, Stephen Dewey, in Prince George County.

After Wythe died, Sweeney was charged with poisoning Wythe and Brown with arsenic. Prominent attorneys william Wirt and Edmund Randolph defended Sweeney. The prosecutor was Philip Norborne Nicholas, Randolph's son-in-law. Early on, the judges quashed the murder charge relating to Brown, because of his race. A jury acquitted Sweeney of Wythe's murder. Some attributed the verdict to the botched autopsy (which failed to use well-known tests for arsenic), and equivocal testimony by the Physicians. Others blamed Virginia law, which since 1732 forbade testimony by black witnesses, whether free or enslaved. In a separate trial for check forgery, Sweeney was convicted. However, that conviction was overruled on appeal based on a technicality in the forgery law that Wythe and Jefferson had drafted years earlier (recognizing the crime only against individual victims, not against corporations such as the bank). Sweeney left for Tennessee. There, he reportedly was later convicted and jailed for stealing a horse. Afterward he disappeared in history.

In October 1748, family connections (Benjamin Waller was related to Zachary Lewis) probably helped Wythe secure his first government job, as clerk to two powerful committees of the House of Burgesses, Privileges and Elections and Propositions and Grievances. Wythe also continued to practice law before those committees and the General Court in Williamsburg, as was permitted at the time. In 1750, Wythe was first elected as one of Williamsburg's aldermen. Wythe also briefly served as the king's attorney general in 1754–1755, appointed by lieutenant governor Robert Dinwiddie while Peyton Randolph traveled to London on the burgesses' behalf to appeal Dinwiddie's charging a one pistole fee to affix an official seal to land patents. Wythe resigned when Randolph returned from his unsuccessful mission. Lieutenant governor Robert Dinwiddie returned to England less than three years after Peyton Randolph's return.

However, Wythe probably continued to live in Williamsburg, for his legislative work continued, and he married Elizabeth Taliaferro. Her father, planter Richard Taliaferro, built a house for them in Williamsburg, which is still called the George Wythe House even though Wythe only had a life estate in the property after Taliaferro's death. Wythe served as Williamsburg's delegate through the sessions of 1754 and 1755 (but not in the sessions of the Assembly of 1756–1758). During that gap, Wythe was reappointed clerk to the committees on Privileges and Elections and Propositions and Grievances, as well as to the Committee for Courts of Justice, and in 1759 to the Committee of Correspondence (with the colony's agent in England). In 1759, The College of william and Mary elected Wythe as its burgess to replace Peyton Randolph, and reelected Wythe in 1760 and 1761. Wythe helped oversee defense expenditures related to the French and Indian War, and later with Richard Henry Lee retirement of the paper money issued to fund the war, which became a scandal in 1766, as discussed below. For the Assemblies of 1761, 1765 and 1767, Wythe was one of the two burgesses representing Elizabeth City County.

Wythe's teaching career began with his appointment in 1761 to the Board of Visitors of the College of william and Mary, and often both overlapped and drew upon his legal and judicial careers. During more than twenty years, Wythe taught many legal apprentices, as well as students at the college. Among the most famous were Future Presidents Thomas Jefferson and James Monroe; Future senators Henry Clay, Littleton Waller Tazewell and John Breckinridge; Future Virginia judges St. George Tucker and Spencer Roane; Future Chief Justice of the Supreme Court, John Marshall; and Future Associate Justice of the Supreme Court, Bushrod Washington. Proficient in Latin and Greek, as well as known for his devotion to books and learning, Wythe initially taught students on a near-individual apprenticeship basis. Especially after Elizabeth's death in 1787, some private pupils boarded at Wythe's home and received daily instruction in classical languages, as well as political philosophy and law. Of all these men, Wythe remained closest to Jefferson, with whom he worked and corresponded many times in the ensuing decades. In their friendship, together the two men read all sorts of other material, from English literary works, to political philosophy, to the ancient sages.

Although known for his modesty and quiet dignity, Wythe eventually gained a radical reputation for his opposition to the Stamp Act of 1765 and later attempts by the British government to regulate the overseas colony. Meanwhile, Wythe maintained close friendships with successive Governors, Francis Fauquier and Norborne Berkeley, 4th Baron Botetourt. Wythe also earned a reputation for personal integrity, which years later led Rev. Lee Massey to call Wythe "the only honest Lawyer I ever knew." Fauquier, Wythe and college professor william Small often socialized together—conversing about philosophy, natural history, languages, history and other matters. In 1762, Small suggested Wythe supervise the legal training of a star student, Thomas Jefferson, which had profound impact that went beyond their lives.

Meanwhile, the publisher of the Virginia Gazette had died. When it became clear in the spring of 1766 that the Stamp Act was not going to be enforced, two printers set up shops in Williamsburg. Both papers thrived in the ensuing controversies. On July 4, Judge Blair explained in a published letter that the judges had relied upon assurances of "three eminent Lawyers" that they could grant Chiswell bail, as well as two depositions that Routledge had run himself on Chiswell's sword, while stressing that the high bail of 6,000 pounds sterling could be recovered should Chiswell fail to show for trial. In response, 'Dikephilos' wrote that his own investigation agreed that Routledge had been drunk, but Chiswell was not, and further that neither deponent favoring Chiswell had witnessed the brawl. He predicted popular violence if the trial unfairly favored the aristocratic defendant. More published letters followed. On August 1, Wythe identified himself in print as one of the consulted lawyers and said his opinion had been limited to the legal bail issue. After Chiswell returned to Williamsburg in September, his attorney John Wayles published the two depositions given to the judges, which proved to be from Wayles himself and the Cumberland undersheriff. Judge Blair was furious when Wayles added a detail to the published copy of his own deposition (which he had gotten from the court records), apparently in order to track the undersheriff's deposition. Judge Byrd, on the other hand, joined with Wayles to demand a libel indictment against Col. Robert Bolling, Jr., claiming Bolling wrote the anonymous criticism of the bailment published on July 11. The grand jury refused, issuing a no-bill instead. However, Wythe's sterling reputation may have been tarnished. When the assembly reconvened, Robert Carter Nicholas was appointed treasurer, Peyton Randolph became speaker, and his brother John Randolph became king's attorney general, a post to which Wythe had aspired. Chiswell died unexpectedly in his Williamsburg home on October 15, before his trial could begin. Jefferson decades later in his Kentucky Resolution echoed the anonymous 'Virginia Gazette' Writer of September 12, 1766, who stated, "Distrust, the parent of security, is a political virtue of unspeakable utility."

Wythe continued his thriving legal practice with Jefferson's assistance. In 1767, Wythe introduced Jefferson to the bar of the General Court, and was himself appointed clerk to the House of Burgesses. The following year, Wythe wrote the colony's London agent to secure copies of the burgesses' complete journals from the colony's founding until 1752, which were supposed to be transmitted annually to the king, secretary of state and Lords of Trade, stressing "it be not made public nor attended with great expense." The secrecy may have related to continuing unrest in Massachusetts against the Townshend Acts or the administrative interregnum between Fauquier's death in March and Botetourt's arrival in October. Wythe also ordered printed journals of the House of Commons and case law books from London. Wythe's social standing remained high, for fellow aldermen elected him Williamsburg's mayor for the 1768 to 1769 term. Fellow parishioners also elected Wythe to the vestry of Bruton Parish Church in 1769.

Wythe for years followed Virginia precedent (including the 1768 case Blackwell v. Wilkinson) as he adjudicated chancery cases treating slaves as property. Slavery matters often went to chancery, because there were no remedies at law. Virginia slave laws also became more severe as Richmond's importance as a slave trading center for points further south continued to increase, and French planters from what became Haiti came to Virginia with thousands of slaves. Wythe authored two legal opinions that attempted to steer Virginia away from the slave-based legal and economic system that entrenched in the early 19th century.

The next royal Governor, John Murray, 4th Earl of Dunmore, brought Wythe and Virginia to and past the brink of revolution. Dunmore arrived in Williamsburg from New York on September 26, 1771. Rumors of his year as the other colony's governor, which included accusations of graft and companions roughing up local judges, soon followed. Although some cheered his military offensive against the Indians (later known as Lord Dunmore's War) as strengthening Virginia's land claims, Wythe, Jefferson and many others soon took offense at Dunmore's haughty personality. Dunmore attempted to govern without the burgesses, but counterfeiting and other money troubles forced him to convene the Assembly in early 1773. Delegates began by voicing concerns that suspects in the burning of the revenue vessel 'Gaspee' in Rhode Island could be tried in England. When on March 3, 1773 they resolved to establish a Committee of Correspondence, Dunmore prorogued (postponed) the assembly. Moreover, Parliament passed the Tea Act in May, 1773 and on December 16, the Sons of Liberty had the Boston Tea Party. Dunmore tried to reconvene the delegates the following May. On May 24, 1774, the House of Burgesses passed a resolution declaring June 1 as a day of fasting and prayer, which resolution Wythe signed and posted. Enraged, Dunmore dissolved the assembly. The delegates moved to conduct their Business at the Raleigh Tavern, and meet again mid-March in Richmond.

On May 10, 1775, the Second Continental Congress convened in Philadelphia. When war seemed inevitable, Wythe was elected as Virginia's delegate to replace George Washington, who took command of the continental forces. George and Elizabeth Wythe moved to Philadelphia by September, and were inoculated against smallpox, as were fellow delegate Francis Lightfoot Lee and his lady and others. By October, Thomas Jefferson had rejoined the Congress to work with his former Teacher and the other delegates, although personal tragedy forced him to leave for five months in the winter and spring. Wythe accepted many assignments relating to military, currency and other matters. He, John Dickinson and John Jay also went to New Jersey that winter and convinced that colony's assembly to maintain a united front. When petitions and other attempts failed to resolve the crisis by the following summer, all while Dunmore's raiders harassed Virginia settlements from its waterways, Wythe moved and then voted in favor of the resolution for independence that Jefferson had drafted upon his return. His fellow Virginia delegates in Philadelphia held Wythe in such esteem, that they left the first space open for Wythe when they signed the Declaration of Independence. Moreover, John Adams, who did not like many Virginians, thought so highly of Wythe that he wrote 'Thoughts on Government' concerning establishing constitutions for state governments after the war. Earlier in the session, Wythe also exchanged humorous verses with his friend delegate william Ellery of Rhode Island despite their political differences. Wythe thus signed the Declaration of Independence upon his (and his wife's) return to Philadelphia in September. The signers' names were not made public until the following January, for all knew the declaration was an act of treason, punishable by death should their rebellion fail.

Hurrying back to Virginia from Philadelphia, on June 23, 1776, Wythe began helping Virginia establish its new state government. Virginia's constitutional convention had begun months earlier (and had voted on May 15 to instruct its federal delegates to move toward a declaration of independence). Despite his late arrival, Wythe served on a committee with George Mason which jointly designed the Seal of Virginia, inscribed with the motto Sic Semper Tyrannis, which remains in use today. The reverse side shows three Roman goddesses, Libertas surrounded by Ceres and Aeternitas. Wythe's most noteworthy contributions in establishing the new state government began when he again returned from Philadelphia that winter. Wythe served on a committee with Thomas Jefferson and Edmund Pendleton to revise and codify its laws, and also helped establish the new state court system. Although few of their 100+ separate proposed bills were ever passed, some concepts such as religious freedom, public records access, and education became important concepts in the new republic, as did the concept of intermediate appellate courts. When a fall incapacitated Pendleton, Jefferson and Wythe redrafted his portion (much to Pendleton's dismay). Wythe also replaced Pendleton as Speaker of the Virginia House of Delegates the following term (1777–1778).

In 1777, Wythe became one of the three judges on the new High Court of Chancery. Administering equity and developing that branch of the law became his mission for the rest of his life. Wythe was elected to serve as a federal judge on the Court of Appeals in Cases of Capture in 1780, but declined to serve. Wythe particularly despised lawyers who protracted litigation at great cost to the parties, though to their own benefit, and even in his last days regretted the burden delays placed upon those seeking justice from his court. In the judicial reorganization of 1788, Wythe became sole Judge of the Chancery Court of Virginia, refusing to be promoted with Edmund Pendleton to the Supreme Court of Appeals (now known as the Virginia Supreme Court). Both men refused offers from President George Washington of judgships on the new federal courts, although their colleague John Blair accepted an appointment to the United States Supreme Court. In 1802, the legislature created two more territorial Chancery Courts, but Wythe remained in Richmond.

In 1779, Governor Jefferson appointed Wythe to the newly created Chair of Law and Police, making Wythe the first law professor in the United States. As a law professor, Wythe introduced a lecture system based on the Commentaries published by william Blackstone, as well as Matthew Bacon's New Abridgement of the Law, and Acts of Virginia's Assembly. Wythe also developed experiential tools, including moot courts and mock legislative sessions, tools that are still used today. However, apprenticeship remained the main mode of learning law in that era, followed by examination before several practicing lawyers. Thus, not only did Marshall and Monroe attend Wythe's lectures for a time, they also affiliated themselves with more experienced lawyers before being admitted to the bar. The college suspended classes during the later days of the revolutionary war, after which Wythe both taught in Williamsburg and performed his duties as judge (mostly in Richmond as the new capital) until the 1788–1789 term. Wythe then resigned from the college and announced that he planned to move to Richmond to concentrate on his judicial duties. Travel to the new capital for the four judicial sessions each year may have become onerous, many of Wythe's friends and colleagues had died or moved, and Williamsburg's intellectual and cultural life had also declined after the state capital moved upriver. Litigation involving professor Rev. John Bracken also distressed Wythe.

In the 1782 Commonwealth v. Caton opinion, Wythe upheld judicial review of legislative actions, in what became a predecessor to Justice Marshall's decision in Marbury v. Madison. After Virginia courts had convicted Caton and two other men of treason, they appealed to the legislature for pardons. The House of Delegates approved their pardon request, but the state Senate refused. Wythe decided that not only did the court have the right to review that pardon, the judiciary was obliged to "say to them, here is the limit of your authority; and hither, shall you go, but no further." Pendleton and Blair agreed with this principle of judicial review, although on slightly different grounds. Nonetheless, after the decision, both legislative houses pardoned the men, sparing them execution.

One scholar states, without extensive documentation, that the Problem of slavery preoccupied Wythe in his last years. In 1785, Jefferson assured English abolitionist Richard Price that Wythe's sentiments against slavery were unequivocal. During the first two decades after the war, so many Virginians freed slaves that the percentage of free blacks in the state rose from less than 1 percent to nearly 10 percent by 1810. However, this era also saw the development of increasingly harsh slave laws, particularly as slaveholders feared rebellions similar to the Haitian Revolution which began in 1791. Tensions increased in 1793 when 137 vessels bearing French refugees from Haiti (and their slaves) arrived in Richmond. John Marshall and other prominent citizens wrote Governor Henry Lee III about slave rebellion rumors, and white militia armed. Also, invention of the cotton gin in 1794 made cotton production using slave labor particularly profitable in the lower south, and those planters imported slaves from Maryland and Virginia, especially when importing slaves from Africa and Britain's Caribbean colonies became illegal and difficult. In 1798, Virginian legislators forbad members of slave emancipation societies from serving on juries involving slave freedom suits, which probably affected Wythe as a judge in such cases, as well as led the Virginia Abolition Society to all but disappear in that year. In 1800 Governor James Monroe called out troops that crushed the rebellion led by Gabriel Prosser near Richmond, and 35 slaves were executed. Further rumors of slave insurrections led to alarms and executions, sometimes without judicial process, in 1802 and 1803. Also, in the legislative session that began in the spring of 1806, the year of Wythe's death, a law passed requiring freed slaves to leave the state within 12 months, although a later modification allowed local courts to allow certain manumitted slaves to remain.

When Elizabeth died on August 18, 1787, Wythe returned some slaves whom her father had bequeathed to Elizabeth to her remaining relatives. Wythe filed manumission papers for his long-time housemaid and cook Lydia Broadnax on August 20, 1787, two days after Elizabeth's death. Four years later, Lydia accompanied Wythe as he moved to Richmond, where he had previously commuted four times yearly to deal with Chancery Court Business. In addition, a young mixed-race youth, Michael Brown, born free in 1790, lived in Wythe's household. By 1797 Broadnax owned her own home, where she and Brown lived, and took in boarders. Wythe had taken an interest in Brown, taught him Greek and shared his library with him. On January 29, 1797, Wythe also freed Benjamin, another adult slave who continued to work as his servant in Richmond; Wythe also named Benjamin a beneficiary in his 1803 will, which also included money for Brown's continued education.

In 1790 Wythe received the honorary degree of LL.D. from the College of william & Mary.

However, Chancellor Wythe's decisions were modified or overruled many times, particularly by the appeals court that his former student Spencer Roane joined in 1794. In 1795, Wythe published analyses of some of his cases and subsequent appellate decisions, and added a few more pamphlets later. Although this publication offended Pendleton, he decided against replying in kind. One of Wythe's most famous decisions (unpopular at the time), Page v. Pendleton, upheld the authority of the 1783 federal peace treaty with Great Britain which required debts to British merchants be paid under the contract terms, although Virginia had passed a law (used by Edmund Pendleton and other executors of the will of Speaker John Robinson discussed above) that allowed payment in depreciated paper currency. In Roane v. Innes, Wythe upheld revolutionary war soldiers's pension claims, but was reversed. Pendleton died in 1803, just before he could deliver an opinion attempting to reverse Wythe in Turpin v. Lockett, which dealt with the sale of the disestablished church's glebe lands, nominally at least to support the poor.

Wythe and Pendleton both sat on the Chancery court bench which granted freedom to slaves in Pleasants v. Pleasants. However, that decision was appealed, and in 1799, after Virginia passed a law forbidding abolitionists from serving on juries in freedom suits, Wythe's decision was modified by the appellate court led by Pendleton and Roane. This case concerned a Quaker's 1771 will, which purported to free slaves before Wythe and Jefferson drafted the 1782 law which legalized manumission in Virginia. Robert Pleasants and some of his siblings had freed about 100 slaves as his late father had requested, after that became legal and they became 30, as the will specified. Robert Pleasants also lobbied extensively for manumission laws and founded the Virginia Abolition Society in 1790. John Marshall and John Warden represented the slaves seeking their freedom, and Pleasants as executor of his father's will, as they jointly sued the siblings who failed to obey the testamentary instruction. Each justice on the Court of Appeals in 'Pleasants v. Pleasants,' 2 Call 338–339 (1799) agreed with Wythe that the will could be enforced and called the slaves free. However, none of the justices (all slaveholders) thought Wythe's grant of backpay proper, and they all also agreed that children borne to slave parents would not actually gain their freedom until they repaid their owners' expenses in raising them, which in the intervening decades became quite large. Thus, although John Pleasants died holding over 530 slaves, fewer than a quarter received their freedom.

In Richmond, Wythe continued his pursuit of knowledge, and even began learning Hebrew from Rabbi Seixas. One of Wythe's last pupils, william Munford, called Wythe "one of the most remarkable men I ever knew" and particularly remembered Wythe's scholarly philosophy, "Don't skim it; read deeply, and ponder what you read; they begin to make lawyers now without the 'biginti annorum lucubrationes' (twenty years of reflection) of Lord Coke; they are mere skimmers of the law, and know little else." St. George Tucker, his one time student and fellow judge, succeeded Wythe as the college's law professor, and published an annotated edition of Blackstone's work before also resigning in 1804 to succeed Edmund Pendleton on Virginia's Supreme Court of Appeals. In 1920, the college (now a university) established a law school, which it named after Wythe and Marshall.

By 1805, Wythe's sister's grandson, 17-year-old George Wythe Sweeney, had come to live with his elderly namesake. The following spring, Wythe realized Sweeney stole some of his books, probably to repay gambling debts and support a dissolute lifestyle. Wythe also revised his will in early 1806 because Thomas Jefferson had agreed to educate the young mulatto, although those new provisions would have no effect if Brown died before Sweeney, as happened.

On Pentecost Sunday, May 25, Wythe, Broadnax and Brown all became violently ill. Richmond's leading doctors, Wythe's old friend James McClurg, James McCaw and his personal physician william Foushee at first suspected cholera, dismissing Wythe's claims of being poisoned. Two days later, Sweeney tried to cash a $100 check drawn on Wythe's account, which the bank found suspicious because Wythe's illness had become news throughout the city. The bank retrieved several earlier checks, which Wythe had previously denied signing. Gravely ill but still trying to work on legal matters, Wythe refused to post bail for Sweeney, who was jailed. Upon hearing that Brown had died on June 1, Wythe signed a codicil to his will drafted by Edmund Randolph that disinherited George Sweeney in favor of Charles, Jane and Ann Sweeney. Wythe also told the doctors "cut me." Although McClurg often used bloodletting, the doctors agreed that Wythe actually called for an autopsy after his death, which happened on June 8, 1806. Oddly, Houlder Hudgins served as administrator for at least two of the siblings, who soon became Wythe's heirs.

In his will, Wythe left his large book collection to Thomas Jefferson. This was part of the collection which Jefferson later sold to create the Library of Congress. Jefferson praised Wythe as "my ancient master, my earliest and best friend, and to him I am indebted for first impressions which have [been] the most salutary on the course of my life." However, Jefferson later refused an offer of Wythe's lecture notes and other legal papers, believing they should go instead to what became the Library of Virginia. Last reported either in the possession of Spencer Roane (who burned many papers before his own death) or his ally Thomas Ritchie (publisher of the Richmond Enquirer), they were reported lost by the 1830s.

The actor william Bakewell played Wythe in the 1965 episode entitled "George Mason" of the NBC documentary series, Profiles in Courage, based on writings of John F. Kennedy. Laurence Naismith portrayed Mason, and Arthur Franz played James Madison.