Thurloe was born in Essex in 1616 and was baptised on June 12. His father was Thomas Thurloe, rector of Abbot's Roding. He was trained as a lawyer in Lincoln's Inn. He was first in the service of Oliver St. John, and, in January 1645, became a secretary to the parliamentary commissioners at Uxbridge. In 1647 Thurloe was admitted to Lincoln's Inn as a member. He remained on the sidelines during the English Civil War but after the accession of Oliver Cromwell, became part of his government in 1652 he was named a secretary for state.
In 1653 he became head of intelligence and developed a widespread network of spies in England and on the continent. These included the Dutch diplomat and historian Lieuwe van Aitzema, the mathematician John Wallis, who established a code-breaking department, and diplomat and mathematician Samuel Morland, who served as Thurloe's assistant. Thurloe's service broke the Sealed Knot, a secret society of Royalists and uncovered various other plots against the Protectorate. In 1654 he was elected to Parliament as the member for Ely. He supported the idea that Cromwell should adopt a royal title.
In 1656 Thurloe took charge of the post office. His spies were then able to intercept mail, and he exposed Edward Sexby's 1657 plot to assassinate Cromwell and captured would-be assassin Miles Sindercombe and his group. (Ironically, Thurloe's own department was also infiltrated: in 1659 Morland became a Royalist agent and alleged that Thurloe, Richard Cromwell and Sir Richard Willis - a Sealed Knot member turned Cromwell agent - were plotting to kill the future King Charles II.)
In 1657 Thurloe became a member of Cromwell's second council, as well as governor of the London Charterhouse school, and in 1658 he became chancellor of the University of Glasgow. After the death of Oliver Cromwell in 1658, he supported his son Richard Cromwell as Lord Protector and, in 1659, represented Cambridge University in the Third Protectorate Parliament. Later that year various parties accused him of arbitrary decisions as head of intelligence, and he was deprived of his offices. Reinstated as a secretary of state in February 27 1660, he resisted the return of Charles II.
After the Restoration, Thurloe was arrested for high treason on May 15, 1660, but was not tried. He was released on June 29 on the condition that he would assist the new government upon request. He retired from public life but served as a behind-the-scenes authority on foreign affairs and wrote informative papers for Edward Hyde, 1st Earl of Clarendon, but he did not become part of any new government.
John Thurloe died in February 21, 1668 in his chambers in Lincoln's Inn and was buried in the chapel. His correspondence is kept in the Bodleian Library, Oxford and in the British Museum. Thomas Birch published part of it in 1742.