In 1651 Sancroft was dismissed as a fellow at the University of Cambridge for refusing to take the Oath of Engagement, a declaration to uphold the government of the Commonwealth. After the Restoration of King Charles II (1660), he was made a royal chaplain, and from 1664 to 1677 he served as dean of St. Paul's Cathedral in London. In 1678 he became archbishop of Canterbury.

Sancroft, however, felt he could not remain silent in the face of what he considered attempts by King James II (reigned 1685–88) to undermine the Anglican church. With six other bishops he petitioned James for permission not to order his clergy to commend James's second Declaration of Indulgence (April 1688), which suspended religious and civil restrictions against Roman Catholics and Protestant dissenters. Because the restrictions had been imposed by parliamentary statute, Sancroft alleged that the declaration was unconstitutional. James responded by imprisoning the bishops in the Tower of London and bringing them to trial on charges of seditious libel. Their acquittal (June 30) was greeted with widespread popular rejoicing. After James was overthrown by William of Orange (William III) in November–December 1688, Sancroft—despite his opposition to James—rejected William's claim to the throne. Along with a number of other Anglican clergymen (the so-called “Nonjurors (Nonjuror)”), he refused to take the oaths of allegiance to William that Parliament had approved. Consequently, he was deprived of his bishopric in 1690.