Weelkes was baptised in the little village church of Elsted in Sussex on 25 October 1576. It has been suggested that his father was John Weeke, rector of Elsted, although there is no documentary evidence of the relationship. In 1597 his first volume of madrigals was published, the preface noting that he was a very young man when they were written; this helps to fix the date of his birth to somewhere in the middle of the 1570s. Early in his life he was in service at the house of the courtier Edward Darcye. At the end of 1598, at the probable age of 22, Weelkes was appointed organist at Winchester College, where he remained for two or three years, receiving the salary of 13s 4d per quarter. His remuneration included board and lodging.

During his Winchester period, Weelkes composed a further two volumes of madrigals (1598, 1600). He obtained his B. Mus. Degree from New College, Oxford in 1602, and moved to Chichester to take up the position of organist and informator choristarum (instructor of the choristers) at the Cathedral at some time between October 1601 and October 1602. He was also given a lay clerkship at the Cathedral, being paid £15 2s 4d annually alongside his board, lodging and other amenities. The following year he married Elizabeth Sandham, from a wealthy local family. They had three children and it was rumored that Elizabeth was already pregnant at the time of the marriage.

Weelkes' fourth and final volume of madrigals, published in 1608, carries a title page where he refers to himself as a Gentleman of the Chapel Royal; however, records at the Chapel Royal itself do not mention him, so at most he could only have been a Gentleman Extraordinary - one of those who were asked to stand in until a permanent replacement was found.

Weelkes was later to find himself in trouble with the Chichester Cathedral authorities for his heavy drinking and immoderate behavior. In 1609 he was charged with unauthorized absence, but no mention of drunken behavior is made until 1613, and J Shepherd, a Weelkes scholar, has suggested caution in assuming that his decline began before this date. In 1616 he was reported to the Bishop for being 'noted and famed for a common drunkard and notorious swearer & blasphemer'. The Dean and Chapter dismissed him for being drunk at the organ and using bad language during divine service. He was however reinstated and remained in the post until his death, although his behavior did not improve; in 1619 Weelkes was again reported to the Bishop:

Dyvers times & very often come so disguised either from the Tavern or Ale house into the quire as is much to be lamented, for in these humors he will both curse & swear most dreadfully, & so profane the service of God ... and though he hath been often times admonished ... to refrain these humors and reform himself, yet he daily continues the same, & is rather worse than better therein.

In 1622 Elizabeth Weelkes died. Thomas Weelkes was, by this time, reinstated at Chichester Cathedral, but appeared to be spending a great deal of time in London. He died in London in 1623, in the house of a friend, almost certainly on 30 November and was buried on 1 December 1623 at St Bride's Fleet Street. Weelkes' will, made the day before he died at the house of his friend Henry Drinkwater of St Bride's parish, left his estate to be shared between his three children, with a large 50s legacy left to Drinkwater for his meat, drink and lodging. He has a memorial stone in Chichester Cathedral.