Tyndale began a Bachelor of Arts degree at Magdalen Hall (later Hertford College) of Oxford University in 1506 and received his B.A. in 1512, the same year becoming a subdeacon. He was made Master of Arts in July 1515 and was held to be a man of virtuous disposition, leading an unblemished life. The M.A. allowed him to start studying theology, but the official course did not include the systematic study of Scripture. As Tyndale later complained:

He was a gifted Linguist and became fluent over the years in French, Greek, Hebrew, German, Italian, Latin, and Spanish, in addition to English. Between 1517 and 1521, he went to the University of Cambridge. Erasmus had been the leading Teacher of Greek there from August 1511 to January 1512, but not during Tyndale's time at the university.

Tyndale became chaplain at the home of Sir John Walsh at Little Sodbury and tutor to his children around 1521. His opinions proved controversial to fellow clergymen, and the next year he was summoned before John Bell, the Chancellor of the Diocese of Worcester, although no formal charges were laid at the time. John Foxe describes an argument with a "learned" but "blasphemous" clergyman which occurred after the harsh meeting with Bell and other church Leaders, and near the end of Tyndale's time at Little Sodbury. The clergyman asserted to Tyndale, "We had better be without God's laws than the Pope's." Tyndale responded: "I defy the Pope, and all his laws; and if God spares my life, ere many years, I will cause the boy that driveth the plow to know more of the Scriptures than thou dost!"

While translating, Tyndale followed Erasmus' (1522) Greek edition of the New Testament. In his preface to his 1534 New Testament ("WT unto the Reader"), he not only goes into some detail about the Greek tenses but also points out that there is often a Hebrew idiom underlying the Greek. The Tyndale Society adduces much further evidence to show that his translations were made directly from the original Hebrew and Greek sources he had at his disposal. For Example, the Prolegomena in Mombert's William Tyndale's Five Books of Moses show that Tyndale's Pentateuch is a translation of the Hebrew original. His translation also drew on the Latin Vulgate and Luther's 1521 September Testament.

Tyndale left for London in 1523 to seek permission to translate the Bible into English. He requested help from Bishop Cuthbert Tunstall, a well-known classicist who had praised Erasmus after working together with him on a Greek New Testament. The bishop, however, declined to extend his patronage, telling Tyndale that he had no room for him in his household. Tyndale preached and studied "at his book" in London for some time, relying on the help of cloth merchant, Humphrey Monmouth. During this time, he lectured widely, including at St Dunstan-in-the-West.

Tyndale left England and landed on continental Europe, perhaps at Hamburg, in the spring of 1524, possibly travelling on to Wittenberg. There is an entry in the matriculation registers of the University of Wittenberg of the name "Guillelmus Daltici ex Anglia", and this has been taken to be a Latinisation of "William Tyndale from England". He began translating the New Testament at this time, possibly in Wittenberg, completing it in 1525 with assistance from Observant friar william Roy.

In response to allegations of inaccuracies in his translation in the New Testament, Tyndale in the Prologue to his 1525 translation wrote that he never intentionally altered or misrepresented any of the Bible, but that he had sought to "interpret the sense of the scripture and the meaning of the spirit."

Of the first (1526) edition of Tyndale's New Testament only three copies survive. The only complete copy is part of the Bible Collection of Württembergische Landesbibliothek, Stuttgart. The copy of the British Library is almost complete, lacking only the title page and list of contents. Another rarity is Tyndale's Pentateuch, of which only nine remain.

It is possible that Tyndale intended to carry on his work from Hamburg in about 1529. He revised his New Testament and began translating the Old Testament and writing various treatises.

In 1530, he wrote The Practyse of Prelates, opposing Henry VIII's planned annulment of his marriage to Catherine of Aragon in favour of Anne Boleyn, on the grounds that it was unscriptural and that it was a plot by Cardinal Wolsey to get Henry entangled in the papal courts of Pope Clement VII. The king's wrath was aimed at Tyndale. Henry asked Emperor Charles V to have the Writer apprehended and returned to England under the terms of the Treaty of Cambrai; however, the Emperor responded that formal evidence was required before extradition. Tyndale developed his case in An Answer unto Sir Thomas More's Dialogue.

A copy of Tyndale's The Obedience of a Christian Man fell into the hands of Henry VIII, providing the king with the rationale to break the Church in England from the Roman Catholic Church in 1534. In 1535, Tyndale was arrested and jailed in the castle of Vilvoorde (Filford) outside Brussels for over a year. In 1536, he was convicted of heresy and executed by strangulation, after which his body was burnt at the stake. His dying prayer was that the King of England's eyes would be opened; this seemed to find its fulfilment just two years later with Henry's authorisation of the Great Bible for the Church of England, which was largely Tyndale's own work - missing sections supplemented with translations by Miles Coverdale. Hence, the Tyndale Bible, as it was known, continued to play a key role in spreading Reformation ideas across the English-speaking world and, eventually, to the British Empire.

Eventually, Tyndale was betrayed by Henry Phillips to the imperial authorities, seized in Antwerp in 1535, and held in the castle of Vilvoorde (Filford) near Brussels. He was tried on a charge of heresy in 1536 and was condemned to be burned to death, despite Thomas Cromwell's intercession on his behalf. Tyndale "was strangled to death while tied at the stake, and then his dead body was burned". His final words, spoken "at the stake with a fervent zeal, and a loud voice", were reported as "Lord! Open the King of England's eyes." The traditional date of commemoration is 6 October, but records of Tyndale's imprisonment suggest that the actual date of his execution might have been some weeks earlier. Foxe gives 6 October as the date of commemoration (left-hand date column), but gives no date of death (right-hand date column). Biographer David Daniell states the date of death only as "one of the first days of October 1536".

In 1611, the 54 scholars who produced the King James Bible drew significantly from Tyndale, as well as from translations that descended from his. One estimate suggests that the New Testament in the King James Version is 83% Tyndale's and the Old Testament 76%. His translation of the Bible was the first to be printed in English, and became a model for subsequent English translations; in 2002, Tyndale was placed at number 26 in the BBC's poll of the 100 Greatest Britons.

The Tyndale Monument was built in 1866 on a hill above his supposed birthplace, North Nibley, Gloucestershire.

A bronze statue by Sir Joseph Boehm commemorating the life and work of Tyndale was erected in Victoria Embankment Gardens on the Thames Embankment, London in 1884. It shows his right hand on an open Bible, which is itself resting on an early printing press.

A stained-glass window commemorating Tyndale was made in 1911 for the British and Foreign Bible Society by James Powell and Sons. In 1994, when the Society moved their offices, the window was reinstalled in the chapel of Hertford College. Tyndale was at Magdalen Hall, Oxford, which became Hertford College in 1874. The window depicts a full-length portrait of Tyndale, a cameo of a printing shop in action, some words of Tyndale, the opening words of Genesis in Hebrew, the opening words of John's Gospel in Greek, and the names of other pioneering Bible translators. The portrait is based on the oil painting that hangs in the college's dining hall. A stained glass window by Arnold Robinson in Tyndale Baptist Church, Bristol also commemorates the life of Tyndale.

A memorial to Tyndale stands in Vilvoorde, where he was executed. It was erected in 1913 by Friends of the Trinitarian Bible Society of London and the Belgian Bible Society. There is also a small william Tyndale Museum in the town, attached to the Protestant church.

Contention from Roman Catholics came not only from real or perceived errors in translation but also a fear of the erosion of their social power if Christians could read the Bible in their own language. "The Pope's dogma is bloody", Tyndale wrote in The Obedience of a Christian Man. Thomas More (since 1935 in the Roman Catholic Church, Saint Thomas More) commented that searching for errors in the Tyndale Bible was similar to searching for water in the sea, and charged Tyndale's translation of The Obedience of a Christian Man with having about a thousand falsely translated errors. Bishop Tunstall of London declared that there were upwards of 2,000 errors in Tyndale's Bible, having already in 1523 denied Tyndale the permission required under the Constitutions of Oxford (1409), which were still in force, to translate the Bible into English.

The translators of the Revised Standard Version in the 1940s noted that Tyndale's translation inspired the translations that followed, including the Great Bible of 1539, the Geneva Bible of 1560, the Bishops' Bible of 1568, the Douay-Rheims Bible of 1582–1609, and the King James Version of 1611, of which the RSV translators noted: "It [the KJV] kept felicitous phrases and apt expressions, from whatever source, which had stood the test of public usage. It owed most, especially in the New Testament, to Tyndale". Many scholars today believe that such is the case. Moynahan writes: "A complete analysis of the Authorised Version, known down the generations as "the AV" or "the King James" was made in 1998. It shows that Tyndale's words account for 84% of the New Testament and for 75.8% of the Old Testament books that he translated." Joan Bridgman makes the comment in the Contemporary Review that, "He [Tyndale] is the mainly unrecognised translator of the most influential book in the world. Although the Authorised King James Version is ostensibly the production of a learned committee of churchmen, it is mostly cribbed from Tyndale with some reworking of his translation."

By tradition Tyndale's death is commemorated on 6 October. There are commemorations on this date in the church calendars of members of the Anglican Communion, initially as one of the "days of optional devotion" in the American Book of Common Prayer (1979), and a "black-letter day" in the Church of England's Alternative Service Book. The Common Worship that came into use in the Church of England in 2000 provides a collect proper to 6 October, beginning with the words:

A life-sized bronze statue of a seated william Tyndale at work on his translation by Lawrence Holofcener (2000) was placed in the Millennium Square, Bristol, United Kingdom. In 2008, vandals attacked the statue, which was taken away, repaired, and reinstalled.

George Steiner in his book on translation After Babel refers to "the influence of the genius of Tyndale, the greatest of English Bible translators..." [After Babel p. 366]. He has also appeared as a character in two plays dealing with the King James Bible, Howard Brenton's Anne Boleyn (2010) and David Edgar's Written on the Heart (2011).

The hierarchy of the Roman Catholic Church did not approve of some of the words and phrases introduced by Tyndale, such as "overseer", where it would have been understood as "bishop", "elder" for "priest", and "love" rather than "charity". Tyndale, citing Erasmus, contended that the Greek New Testament did not support the traditional Roman Catholic readings. More controversially, Tyndale translated the Greek ekklesia (Greek: εκκλησία), (literally "called out ones") as "congregation" rather than "church". It has been asserted this translation choice "was a direct threat to the Church's ancient—but so Tyndale here made clear, non-scriptural—claim to be the body of Christ on earth. To change these words was to strip the Church hierarchy of its pretensions to be Christ's terrestrial representative, and to award this honour to individual worshippers who made up each congregation."