According to his own account, he entered the Service of the Queen in 1557, a year before her accession to the throne, when they were both in their mid-twenties, but he did not begin his Service of the state until the Northern Rebellion of 1569, when he armed a troop of thirty horses at his own expense. In his early years, too, he had served the 6th Earl of Shrewsbury, but then became one of Leicester's men. There is no foundation for the frequently-repeated assertion that he worked for Burghley and Walsingham. As an independent, self-financed operator with his own squadron of "instruments" as he called them, he worked with both Burghley and Walsingham under commission from the Privy Council, and was on very good terms with them both, though he always considered himself the Queen's personal servant and friend.

He represented Beverley in Parliament in 1572. He would later return to Parliament as MP for Old Sarum in 1584 and 1586.

Topcliffe was a tireless agent of the English government's determination to eradicate Catholicism from England. At the height of his power, from the late 1580s until 1595, he hunted, captured, arrested, and interrogated many prisoners - often with torture. Topcliffe's infamous interrogations took place either in the Tower of London, in Bridewell prison, or even at his own house in Westminster, next to the Gatehouse prison. Exiled Catholic intelligencer Richard Verstegan regularly reported on Topcliffe's activities, condemning him "whose inhuman cruelty is so great, as he will not spare to extend any torture whatsoever". His favorite method (which he may have introduced) was to hang a prisoner by the hands in the gauntlets or manacles, a procedure which, though exquisitely painful, left no permanent injury if properly administered. Improperly administered, it caused permanent internal injury and even death. There is some evidence that he used psychological torture as well, keeping prisoners awake, for instance. Furthermore, Topcliffe frequently used rape as a method of extracting information.

—Saint Eustace White, S.J., written to Father Henry Garnet from prison. 23 November 1591. Quoted in The Other Face; Catholic Life Under Elizabeth I, by Father Philip Caraman, pages 235–236.

Topcliffe's victims included the Jesuits Robert Southwell, and Henry Walpole. Topcliffe features numerous times in John Gerard, S.J.'s autobiography of his days as a hunted priest in Elizabethan England. In it, he is described as, "old and hoary and a veteran in evil". In early 1592, he or one of his men, Nicholas Jones, raped or seduced a prisoner in the Gatehouse prison, Anne Bellamy, and persuaded her to arrange the capture of the Jesuit priest Robert Southwell at her family's house outside London. When Bellamy married Jones, Topcliffe tried to force her father to give the couple an estate. What became of them is unknown, but the Bellamy family was ruined. Anne Bellamy was the Queen's prisoner, and therefore entitled to her protection, yet no action was taken against Topcliffe over this affair, even though the Queen knew about the pregnancy, and knew that Topcliffe had spirited the woman away to his house in Lincolnshire.

In August 1597, the Council commissioned Topcliffe to investigate the play by Ben Jonson and Thomas Nashe, The Isle of Dogs, which upset the authorities so badly. Fortunately for Jonson, by then Topcliffe's general commission had been withdrawn, and his power much curtailed, though he never lost favor with the Queen and the Cecils. It is possible, though unlikely, that he initiated the inquiry.

Topcliffe died in November or December 1604 at the age of about 73. His heir, Charles, succeeded to a heavily encumbered estate. Like Walsingham, Topcliffe impoverished himself by his Service to the crown.

Richard Topcliffe was portrayed by Brian Wilde in the 1971 British television mini-series Elizabeth R. Topcliffe is featured in Rory Clements' "John Shakespeare" murder mystery novel series as a sadistic torturer. Topcliffe is also portrayed as a torturer and persecutor of Catholics in the Sir Robert Carey and James Enys novels by P.F. Chisholm/Patricia Finney.

—Father Henry Garnet, "Account of the Trial of Robert Southwell." Quoted in Caraman's The Other Face, page 230.

Topcliffe is portrayed by Ewen Bremner in the 2017 American television series Will, a fictional look at the life of young william Shakespeare. In the series, Shakespeare bases the scheming title character of Richard III on Topcliffe.