He graduated BD. in 1580 and DD. five years later. In 1585 he was appointed treasurer of St Paul's cathedral, London, and in 1586 was made a member of the ecclesiastical commission. On the 9th of February 1589 he preached at Paul's Cross a sermon, the substance of which was a passionate attack on the Puritans. He described their speeches and proceedings, caricatured their motives, denounced the exercise of the right of private judgment, and set forth the divine right of bishops in such strong language that one of the queen’s councillors held it to amount to a threat against the supremacy of the crown.
In the following year Bancroft was made a prebendary of St Paul's; he had been canon of Westminster since 1587. He was chaplain successively to Lord Chancellor Hatton and Archbishop Whitgift. In June 1597 he was consecrated Bishop of London; and from this time, in consequence of the age and incapacity for business of Archbishop Whitgift, he was virtually invested with the power of primate, and had the sole management of ecclesiastical affairs. Among the more noteworthy cases which fell under his direction were the proceedings against “ Martin Mar-Prelate,” Thomas-Cartwright and his friends, and John Penry, whose “seditious writings” he caused to be intercepted and given up to the lord keeper.
In 1600 he was sent on an embassy, with others, to Embden, for the purpose of settling certain matters in dispute between the English and the Danes. This mission, however, failed. Bancroft was present at the death of Queen Elizabeth. In March 1604 Bancroft, on Whitgift's death, was appointed by royal writ president of convocation then assembled; and he there presented a book of canons collected by himself. It was adopted and received the royal approval, but was strongly opposed and set aside by parliament two months afterwards. In the following November he was elected successor to Whitgift in the see of Canterbury. He continued to show the same zeal and severity as before, and with so much success that Lord Clarendon, writing in his praise, expressed the opinion that "if Bancroft had lived, he would quickly have extinguished all that fire in England which had been kindled at Geneva."
In 1608 he was chosen chancellor of the university of Oxford. One of his latest public acts was a proposal laid before parliament for improving the revenues of the church, and a project for a college of controversial divinity at Chelsea. In the last few months of his life he took part in the discussion about the consecration of certain Scottish bishops, and it was in pursuance of his advice that they were consecrated by several bishops of the English church. By this act were laid the foundations of the Scottish Episcopal church. Bancroft was "the chief overseer" of the authorized version of the Bible. He died at Lambeth Palace on the 2nd of November 1610.