He was born in Cornwall, the second son of his family. He was educated at Oxford, at first at Broadgates Hall. He proceeded B.C.L. on 30 June 1516, and D.C.L. on 23 June 1522. He became, before he quit Oxford, principal of Vine Hall.
Moving to London, Tregonwell began to practise in the court of admiralty, of which he became before 1535 principal judge or commissary-general. Henry VIII employed him on government affairs; he carried out his master's wishes smoothly and with a careful regard to the forms of law. He was a privy councillor as early as 1532. He was a proctor for the king in the divorce case. He took part in diplomatic negotiations in the Netherlands in May 1532, John Hackett and Dr. W. Knight being his companions, to settle commercial disputes. He signed the two treaties of peace of 1534 with Scotland on behalf of England. He also took part in the proceedings against the Carthusians, against Sir Thomas More, and against Anne Boleyn.
Tregonwell's major role in the dissolution of the monasteries lay in taking surrenders. He visited Oxford University in 1535; otherwise his work lay mainly in the south and west of England. He was also employed in the proceedings against the prisoners taken in the Pilgrimage of Grace, and he was important enough for Cromwell to talk about him as a possible master of the rolls. He became a master in chancery in 1539, was chancellor of Wells Cathedral from 1541 to January 1542-3, a commissioner in chancery in 1544, and a commissioner of the great seal in 1550.
He was knighted on 2 October 1553, and was favoured by Queen Mary in spite of his history. He was M.P. for Scarborough in the parliament of October 1553, and, though he held a prebend, there was no question of objecting to his return, doubtless because he was a layman. Alexander Nowell was ejected from parliament, and Tregonwell was one of the committee which sat to consider his case. In 1555 he was a commissioner on imprisoned preachers. He died on 8 or 13 January 1564-5 at Milton Abbas, Dorset, for which, after the dissolution, he had paid £1,000, and was buried in the north aisle under an altar tomb.
He had married, first, a wife named Kellaway, by whom he had no children; secondly, Elizabeth Bruce, who was buried on 17 January 1582, by whom he had, with other children, Thomas, who died during his father's lifetime, and who was the father of John Tregonwell, who succeeded to Sir John's property.