Booth's parents were noted British Shakespearean actor Junius Brutus Booth and his mistress Mary Ann Holmes, who moved to the United States from England in June 1821. They purchased a 150-acre (61 ha) farm near Bel Air, Maryland, where John Wilkes Booth was born in a four-room log house on May 10, 1838, the ninth of ten children. He was named after English radical Politician John Wilkes, a distant relative. Junius' wife Adelaide Delannoy Booth was granted a divorce in 1851 on grounds of adultery, and Holmes legally wed Junius on May 10, 1851, the youth's 13th birthday.

Historian Benjamin Platt Thomas wrote that Booth "won Celebrity with theater-goers by his romantic personal attraction", but that he was "too impatient for hard study" and his "brilliant talents had failed of full development." Author Gene Smith wrote that Booth's acting may not have been as precise as his brother Edwin's, but his strikingly handsome appearance enthralled women. As the 1850s drew to a close, Booth was becoming wealthy as an actor, earning $20,000 a year (equivalent to about $545,000 today).

The same year that Booth's father married Holmes (1851), he built Tudor Hall on the Harford County property as the family's summer home, while also maintaining a winter residence on Exeter Street in Baltimore in the 1840s–1850s.

The editor's introduction of the 1874 memoir of Booth's sister Asia Booth Clarke states that no individual church was preeminent in the Booth household during her childhood. Booth's mother was Episcopalian, and his father was described as a free spirit who was open to the great teachings of all religions. On January 23, 1853, the 14-year-old Booth was baptized at St. Timothy's Protestant Episcopal Church. The Booth family had traditionally been Episcopalian. Clergyman Charles Chiniquy stated that Booth was a convert to Roman Catholicism later in life. Historian Constance Head also declared that Booth was of this religion. She wrote the paper "Insights on John Wilkes Booth from His Sister Asia's Correspondence" (1982) published in the Lincoln Herald, in which she quotes from a letter of Booth's sister Asia Booth Clarke, in which she wrote that her brother was a Roman Catholic. (Asia Booth Clarke's memoir was published after her death.)

By the age of 16, Booth was interested in the theatre and in politics, and he became a delegate from Bel Air to a rally by the Know Nothing Party for Henry Winter Davis, the anti-immigrant party's candidate for Congress in the 1854 elections. Booth aspired to follow in the footsteps of his father and his actor brothers Edwin and Junius Brutus, Jr. He began practicing elocution daily in the woods around Tudor Hall and studying Shakespeare.

Booth made his stage debut at age 17 on August 14, 1855, in the supporting role of the Earl of Richmond in Richard III at Baltimore's Charles Street Theatre. The audience jeered at the inexperienced actor when he missed some of his lines. He also began acting at Baltimore's Holliday Street Theater, owned by John T. Ford, where the Booths had performed frequently. In 1857, Booth joined the stock company of the Arch Street Theatre in Philadelphia, Pennsylvania, where he played for a full season. At his request, he was billed as "J.B. Wilkes", a pseudonym meant to avoid comparison with other members of his famous thespian family. Jim Bishop wrote that Booth "developed into an outrageous scene stealer, but he played his parts with such heightened enthusiasm that the audiences idolized him." In February 1858, he played in Lucrezia Borgia at the Arch Street Theatre. On opening night, he experienced stage fright and stumbled over his line. Instead of introducing himself by saying, "Madame, I am Petruchio Pandolfo", he stammered, "Madame, I am Pondolfio Pet—Pedolfio Pat—Pantuchio Ped—dammit! Who am I?", causing the audience to roar with laughter.

Later that year, Booth played the part of Mohegan Indian Chief Uncas in a play staged in Petersburg, Virginia, and then became a stock company actor at the Richmond Theatre in Virginia, where he became increasingly popular with audiences for his energetic performances. On October 5, 1858, Booth played the part of Horatio in Hamlet, alongside his older brother Edwin in the title role. Afterward, Edwin led the younger Booth to the theatre's footlights and said to the audience, "I think he's done well, don't you?" In response, the audience applauded loudly and cried, "Yes! Yes!" In all, Booth performed in 83 plays in 1858. Among them were william Wallace and Brutus, having as their theme the killing or overthrow of an unjust ruler. Booth said that, of all Shakespearean characters, his favorite role was Brutus, the slayer of a tyrant.

Booth was strongly opposed to the abolitionists who sought to end slavery in the U.S. He attended the hanging of abolitionist leader John Brown on December 2, 1859, who was executed for leading a raid on the Federal armory at Harpers Ferry in present-day West Virginia. Booth had been rehearsing at the Richmond Theatre when he abruptly decided to join the Richmond Grays, a volunteer militia of 1,500 men traveling to Charles Town for Brown's hanging, to guard against an attempt by abolitionists to rescue Brown from the gallows by force. When Brown was hanged without incident, Booth stood in uniform near the scaffold and afterwards expressed great satisfaction with Brown's fate, although he admired the condemned man's bravery in facing death stoically.

As a popular actor in the 1860s, Booth continued to travel extensively to perform in the North and South, and as far west as New Orleans, Louisiana. According to his sister Asia, Booth confided to her that he also used his position to smuggle the anti-malarial drug quinine, which was crucial to the lives of residents of the Gulf coast, to the South during his travels there, since it was in short supply due to the Northern blockade.

When the Civil War began on April 12, 1861, Booth was starring in Albany, New York. He was outspoken in his admiration for the South's secession, publicly calling it "heroic." This so enraged local citizens that they demanded that he be banned from the stage for making "treasonable statements". Albany's drama critics were kinder, giving him rave reviews. One called him a genius, praising his acting for "never fail[ing] to delight with his masterly impressions." As the Civil War raged across the divided land in 1862, Booth appeared mostly in Union and border states. In January, he played the title role in Richard III in St. Louis and then made his Chicago debut. In March, he made his first acting appearance in New York City. In May 1862, he made his Boston debut, playing nightly at the Boston Museum in Richard III (May 12, 15 and 23), Romeo and Juliet (May 13), The Robbers (May 14 and 21), Hamlet (May 16), The Apostate (May 19), The Stranger (May 20), and The Lady of Lyons (May 22). Following his performance of Richard III on May 12, the Boston Transcript's review the next day called Booth "the most promising young actor on the American stage".

The Fugitives then continued southward, stopping before dawn on April 15 for treatment of Booth's injured leg at the home of Dr. Samuel Mudd, St. Catharine, 25 miles (40 km) from Washington. Mudd later said that Booth told him the injury occurred when his horse fell. The next day, Booth and Herold arrived at the home of Samuel Cox around 4 a.m. As the two Fugitives hid in the woods nearby, Cox contacted Thomas A. Jones, his foster brother and a Confederate agent in charge of spy operations in the southern Maryland area since 1862. The War Department advertised a $100,000 reward ($1.6 million in 2018 USD) by order of Secretary of War Edwin M. Stanton for information leading to the arrest of Booth and his accomplices, and Federal troops were dispatched to search southern Maryland extensively, following tips reported by Federal intelligence agents to Col. Lafayette Baker.

Booth was pro-Confederate, but his family was divided, like many Marylanders. He was outspoken in his love of the South, and equally outspoken in his hatred of Lincoln. As the Civil War went on, Booth increasingly quarreled with his brother Edwin, who declined to make stage appearances in the South and refused to Listen to John Wilkes' fiercely partisan denunciations of the North and Lincoln. In early 1863, Booth was arrested in St. Louis while on a theatre tour, when he was heard saying that he "wished the President and the whole damned government would go to hell." He was charged with making "treasonous" remarks against the government, but was released when he took an oath of allegiance to the Union and paid a substantial fine.

Lincoln won a landslide re-election in early November 1864 on a platform advocating passage of the 13th Amendment to the U.S. Constitution to abolish slavery altogether, and Booth devoted increasing Energy and money to his kidnap plot. He assembled a loose-knit band of Southern sympathizers, including David Herold, George Atzerodt, Lewis Powell (also known as Lewis Payne or Paine), and John Surratt, a rebel agent. They began to meet routinely at the boarding house of Surratt's mother Mrs. Mary Surratt.

Eight others implicated in Lincoln's assassination were tried by a military tribunal in Washington, D.C. and found guilty on June 30, 1865. Mary Surratt, Lewis Powell, David Herold, and George Atzerodt were hanged in the Old Arsenal Penitentiary on July 7, 1865. Samuel Mudd, Samuel Arnold, and Michael O'Laughlen were sentenced to life imprisonment at Fort Jefferson in Florida's Dry Tortugas. Edmund Spangler was given a six-year term in prison. O'Laughlen died in a yellow fever epidemic there in 1867. The others were eventually pardoned in February 1869 by President Andrew Johnson.

Booth's body was shrouded in a blanket and tied to the side of an old farm wagon for the trip back to Belle Plain. There, his corpse was taken aboard the ironclad USS Montauk and brought to the Washington Navy Yard for identification and an autopsy. The body was identified there as Booth's by more than ten people who knew him. Among the identifying features used to make sure that the man that was killed was Booth was a tattoo on his left hand with his initials J.W.B., and a distinct scar on the back of his neck. The third, fourth, and fifth vertebrae were removed during the autopsy to allow access to the bullet. These bones are still on display at the National Museum of Health and Medicine in Washington, D.C. The body was then buried in a storage room at the Old Penitentiary, later moved to a warehouse at the Washington Arsenal on October 1, 1867. In 1869, the remains were once again identified before being released to the Booth family, where they were buried in the family plot at Green Mount Cemetery in Baltimore, after a burial ceremony conducted by Fleming James, minister of Christ Episcopal Church, in the presence of more than 40 people. Russell Conwell visited homes in the vanquished former Confederate states during this time, and he found that hatred of Lincoln still smoldered. "Photographs of Wilkes Booth, with the last words of great martyrs printed upon its borders ... adorn their drawing rooms".

In response, the Maryland Historical Society published an account in 1913 by Baltimore mayor william M. Pegram, who had viewed Booth's remains upon the casket's arrival at the Weaver funeral home in Baltimore on February 18, 1869, for burial at Green Mount Cemetery. Pegram had known Booth well as a young man; he submitted a sworn statement that the body which he had seen in 1869 was Booth's. Others positively identified this body as Booth at the funeral home, including Booth's mother, brother, and sister, along with his dentist and other Baltimore acquaintances. Earlier, The New York Times had published an account by their reporter in 1911 detailing the burial of Booth's body at the cemetery and those who were witnesses. The rumor periodically revived, as in the 1920s when a corpse was exhibited on a national tour by a carnival promoter and advertised as the "Man Who Shot Lincoln". According to a 1938 article in The Saturday Evening Post, the exhibitor said that he obtained St. Helen's corpse from Bates' widow.

In 1907, Finis L. Bates wrote Escape and Suicide of John Wilkes Booth, contending that a Booth look-alike was mistakenly killed at the Garrett farm while Booth eluded his pursuers. Booth, said Bates, assumed the pseudonym "John St. Helen" and settled on the Paluxy River near Glen Rose, Texas, and later moved to Granbury, Texas. He fell gravely ill and made a deathbed confession that he was the fugitive Assassin, but he then recovered and fled, eventually committing suicide in 1903 in Enid, Oklahoma under the alias "David E. George". By 1913, more than 70,000 copies of the book had been sold, and Bates exhibited St. Helen's mummified body in carnival sideshows.

Forty years later, when the centenary of Lincoln's birth was celebrated in 1909, a border state official reflected on Booth's assassination of Lincoln. "Confederate veterans held public services and gave public expression to the sentiment, that 'had Lincoln lived' the days of reconstruction might have been softened and the era of good feeling ushered in earlier". The majority of Northerners viewed Booth as a MadMan or Monster who murdered the savior of the Union, while in the South, many cursed Booth for bringing upon them the harsh revenge of an incensed North instead of the reconciliation promised by Lincoln. A century later, Goodrich concluded in 2005, "For millions of people, particularly in the South, it would be decades before the impact of the Lincoln assassination began to release its terrible hold on their lives".

Garrett's 11-year-old son Richard was an eyewitness. In later years, he became a Baptist minister and widely lectured on the events of Booth's demise at his family's farm. In 1921, Garrett's lecture was published in the Confederate Veteran as the "True Story of the Capture of John Wilkes Booth." According to his account, Booth and Herold arrived at the Garretts' farm, located on the road to Bowling Green, around 3 p.m. on Monday afternoon. Confederate mail delivery had ceased with the collapse of the Confederate government, he explained, so the Garretts were unaware of Lincoln's assassination. After having dinner with the Garretts that evening, Booth learned of the surrender of Johnston's army, the last Confederate armed force of any size. Its capitulation meant that the Civil War was unquestionably over and Booth's attempt had failed to save the Confederacy by Lincoln's assassination. The Garretts also finally learned of Lincoln's death and the substantial reward for Booth's capture. Booth, said Garrett, displayed no reaction other than to ask if the family would turn in the fugitive should they have the opportunity. Still not aware of their guest's true identity, one of the older Garrett sons averred that they might, if only because they needed the money. The next day, Booth told the Garretts that he intended to reach Mexico, drawing a route on a map of theirs. Biographer Theodore Roscoe said of Garrett's account, "Almost nothing written or testified in respect to the doings of the Fugitives at Garrett's farm can be taken at face value. Nobody knows exactly what Booth said to the Garretts, or they to him." Garrett was present at the time and was more likely to know the facts than later biographers.

While Lincoln's funeral train was in New York City on April 24, Lieutenant Edward P. Doherty was dispatched from Washington at 2 p.m. with a detachment of 26 Union Soldiers from the 16th New York Cavalry Regiment to capture Booth in Virginia, accompanied by Lieutenant Colonel Everton Conger, an intelligence officer assigned by Lafayette Baker. The detachment steamed 70 miles (113 km) down the Potomac River on the boat John S. Ide, landing at Belle Plain, Virginia at 10 p.m. The pursuers crossed the Rappahannock River and tracked Booth and Herold to Richard H. Garrett's farm, just south of Port Royal, Caroline County, Virginia. Booth and Herold had been led to the farm on April 24 by william S. Jett, a former private in the 9th Virginia Cavalry whom they had met before crossing the Rappahannock. The Garretts were unaware of Lincoln's assassination; Booth was introduced to them as "James W. Boyd", a Confederate soldier, they were told, who had been wounded in the battle of Petersburg and was returning home.

Author Francis Wilson was 11 years old at the time of Lincoln's assassination. He wrote an epitaph of Booth in his 1929 book John Wilkes Booth: "In the terrible deed he committed, he was actuated by no thought of monetary gain, but by a self-sacrificing, albeit wholly fanatical devotion to a cause he thought supreme." Others have seen less unselfish motives such as shame, ambition, and sibling rivalry for achievement and fame.

The Lincoln Conspiracy (1977) contended that there was a government plot to conceal Booth's escape, reviving interest in the story and prompting the display of St. Helen's mummified body in Chicago that year. The book sold more than one million copies and was made into a feature film called The Lincoln Conspiracy which was theatrically released in 1977. The 1998 book The Curse of Cain: The Untold Story of John Wilkes Booth contended that Booth had escaped, sought refuge in Japan, and eventually returned to the United States. In 1994 two historians together with several descendants sought a court order for the exhumation of Booth's body at Green Mount Cemetery which was, according to their Lawyer, "intended to prove or disprove longstanding theories on Booth's escape" by conducting a photo-superimposition analysis. The application was blocked by Baltimore Circuit Court Judge Joseph H. H. Kaplan, who cited, among other things, "the unreliability of petitioners' less-than-convincing escape/cover-up theory" as a major factor in his decision. The Maryland Court of Special Appeals upheld the ruling.

Booth then jumped from the President's box to the stage, where he raised his knife and shouted "Sic semper tyrannis" (Latin for "Thus always to tyrants," attributed to Brutus at Caesar's assassination and the Virginia state motto), while others said that he added, "I have done it, the South is avenged!" Various accounts state that Booth injured his leg when his spur snagged a decorative U.S. Treasury Guard flag while leaping to the stage. Historian Michael W. Kauffman questioned this legend in his book American Brutus: John Wilkes Booth and the Lincoln Conspiracies, writing in 2004 that eyewitness accounts of Booth's hurried stage exit made it unlikely that his leg was broken then. Kauffman contends that Booth was injured later that night during his FLIGHT to escape when his horse tripped and fell on him, calling Booth's claim to the contrary an exaggeration to portray his own actions as heroic.

Nora Titone recounts in her book My Thoughts Be Bloody (2010) how the shame and ambition of Junius Brutus Booth's illegitimate actor sons Edwin and John Wilkes eventually spurred them to strive for achievement and acclaim as rivals—Edwin, a Unionist, and John Wilkes, the Assassin of Abraham Lincoln.

In December 2010, descendants of Edwin Booth reported that they obtained permission to exhume the Shakespearean actor's body to obtain DNA samples to compare with a sample of his brother John's DNA to refute the rumor that John had escaped after the assassination. Bree Harvey, a spokesman from the Mount Auburn Cemetery in Cambridge, Massachusetts where Edwin Booth is buried, denied reports that the family had contacted them and requested to exhume Edwin's body. The family hoped to obtain samples of John Wilkes's DNA from remains such as vertebrae stored at the National Museum of Health and Medicine in Maryland. On March 30, 2013, museum spokeswoman Carol Johnson announced that the family's request to extract DNA from the vertebrae had been rejected.