His first known musical appointment was in 1532, as organist of Dover Priory (now Dover College), a Benedictine priory in Kent. His career took him to London, then (probably in the autumn of 1538) to Waltham Abbey, a large Augustinian monastery in Essex which was dissolved in 1540. Tallis was paid off and also acquired a book about music and preserved it; one of the treatises in it, by Leonel Power, prohibits consecutive unisons, fifths, and octaves.

The earliest surviving works by Tallis, Salve intemerata virgo, Ave rosa sine spinis and Ave Dei patris filia are devotional antiphons to the Virgin Mary, which were sung in the evening after the last Service of the day and were cultivated in England until at least the early 1540s. Henry VIII's break repudiation of the authority of the Pope over the Catholic Church in 1534 (Henry did not repudiate the doctrines of the Church) and the rise of Thomas Cranmer noticeably influenced the style of music written. Cranmer recommended a syllabic style of music (which is a setting of text where each syllable is sung to one pitch), as his instructions for the setting of the 1544 English Litany make clear. As a result, the writing of Tallis and his contemporaries became less florid. Tallis's Mass for Four Voices is marked with tendencies toward a syllabic and chordal (consisting of or emphasising chords) style and a diminished use of melisma. Tallis provides a rhythmic variety and differentiation of moods depending on the meaning of his texts. Tallis helped found a relationship that was specific to the combining of words and music.

Tallis's next post was at Canterbury Cathedral. He was next sent to Court as Gentleman of the Chapel Royal in 1543 (which later became a Protestant establishment), where he composed and performed for Henry VIII, Edward VI (1547–1553), Queen Mary (1553–1558), and Queen Elizabeth I (1558 until Tallis died in 1585).

The reformed Anglican liturgy was inaugurated during the short reign of Edward VI (1547–53), and Tallis was one of the first church Musicians to write anthems set to English words, although Latin continued to be used alongside the vernacular. The Catholic Mary Tudor set about undoing some of the religious reforms of the preceding decades. Following her accession in 1553, the Roman Rite was restored and compositional style reverted to the elaborate writing prevalent early in the century. Two of Tallis's major works, Gaude gloriosa Dei Mater and the Christmas Mass Puer natus est nobis are believed to be from this period. Puer natus est nobis, based on the Introit for the third Mass for Christmas Day, was perhaps sung at Christmas 1554 when Mary believed she was expecting a male heir. As was the prevailing practice, these pieces were intended to exalt the image of the Queen as well as to praise the Mother of God.

Tallis married around 1552; his wife, Joan, outlived him by four years. They apparently had no children. Late in his life he lived in Greenwich, possibly close to the royal palace: a local tradition holds that he lived on Stockwell Street.

Some of Tallis's works were compiled by Thomas Mulliner in a manuscript copybook called Mulliner Book before Queen Elizabeth's reign, and may have been used by the Queen herself when she was younger. Elizabeth succeeded her half-sister in 1558, and the Act of Uniformity, actually passed in the following year, abolished the Roman Liturgy and firmly established the Book of Common Prayer. Composers resumed writing English anthems, although the practice of setting Latin texts continued among composers employed by Elizabeth's Chapel Royal.

The religious authorities in the beginning of Elizabeth's reign inclined towards Calvinism, which tended to discourage polyphony in church unless the words were clearly audible, or as the 1559 Injunctions stated, 'playnelye understanded, as if it were read without singing'. Tallis wrote nine psalm chant tunes for four voices for Archbishop Parker's Psalter, published in 1567. One of the nine tunes, the "Third Mode Melody", inspired the composition of Fantasia on a Theme of Thomas Tallis by Ralph Vaughan Williams in 1910. His setting of Psalm 67 became known as "Tallis's Canon" and the setting by Thomas Ravenscroft is an adaptation for the hymn "All praise to thee, my God, this night" (1709) by Thomas Ken. As a result of its widespread use in church services, it has become his best-known composition. Meanwhile, however, the Injunctions also allowed a more elaborate piece of music to be sung in church at certain times of the day, and many of Tallis's more complex Elizabethan anthems may have been sung in this context, or alternatively by the many families that sang sacred polyphony at home. Tallis's better-known works from the Elizabethan years include his settings of the Lamentations (of Jeremiah the Prophet) for the Holy Week services and the unique motet Spem in alium written for eight five-voice choirs, for which he is mostly remembered. Though they are often overlooked, he also produced compositions for other monarchs, and several of Tallis's anthems written in Edward's reign, such as his If ye love me, are judged to be on the same level as his Elizabethan works. This oversight stems in part from our incomplete records of his works from previous periods; eleven of eighteen Latin-texted pieces by Tallis from Elizabeth's reign were published, "which ensured their survival in a way not available to the earlier material".

Queen Mary granted Tallis a lease on a manor in Kent that provided a comfortable annual income. In 1575, Queen Elizabeth granted to him and william Byrd a 21-year monopoly for polyphonic music and a patent to print and publish music, which was one of the first arrangements of that type in the country. Tallis's monopoly covered 'set songe or songes in parts', and he composed in English, Latin, French, Italian, or other tongues as long as they served for music in the Church or chamber. Tallis had exclusive rights to print any music, in any language. He and william Byrd were the only ones allowed to use the paper that was used in printing music. Tallis and Byrd used their monopoly to produce Cantiones quae ab argumento sacrae vocantur in 1575, but the collection did not sell well and they appealed to Queen Elizabeth for her support. People were naturally wary of their new publications, and it certainly did not help their case that they were both avowed Roman Catholics. Not only that, they were strictly forbidden to sell any imported music. "We straightly by the same forbid...to be brought out of any forren Realmes...any songe or songes made and printed in any foreen countrie." Also, Byrd and Tallis were not given "the rights to music type fonts, printing patents were not under their command, and they didn't actually own a printing press."

Thomas Tallis died peacefully in his house in Greenwich in November 1585; most historians agree that he died on 23 November, though one source gives the date as 20 November. He was buried in the chancel of the parish of St Alfege Church in Greenwich. To this day, the exact location in St Alfege Church of Tallis's remains is unknown. His remains may have been discarded by labourers between 1712 and 1714, when the church was rebuilt. Nothing remains of Tallis's original memorial in the church. Strype is said in 1720 to have found a brass plate with an engraving on it, which read:

A fictionalized Thomas Tallis was portrayed by Joe Van Moyland in 2007 on the Showtime television series The Tudors.

Little is known about Tallis's early life, but there seems to be agreement that he was born in the early 16th century, toward the close of the reign of Henry VII. The name "Tallis" is derived from the French word "taillis," which means a "thicket."