Randolph was born in Virginia, at Tazewell Hall in Williamsburg. He attended the College of William and Mary, and later studied law at the Inns of Court in London, becoming a member of the bar in 1743. He then returned to Williamsburg and was appointed Attorney General of the Virginia colony the next year.
He served several terms in the Virginia House of Burgesses, the first one beginning in 1748. It was his dual roles as attorney general and as burgess that would lead to an extraordinary conflict of interest in 1751.
The new governor, Robert Dinwiddie, had imposed a fee for the certification of land patents, which was strongly objected to by the House of Burgesses. The House selected Peyton Randolph to represent their cause to Crown authorities in London. In his role as attorney general, though, it was his responsibility to defend actions taken by the governor. Randolph left for London, over the objections of Governor Dinwiddie, and was replaced for a short time as attorney general, but was reinstated on his return at the behest of officials in London, who recommended the Governor drop the new fee, as well.
In 1765 Randolph found himself at odds with a freshman burgess, Patrick Henry, over the matter of how to respond to the Stamp Act. Randolph had been appointed by the House to draft objections to the act, but his more conservative plan was trumped when Henry obtained passage of five of his seven Virginia Stamp Act Resolutions. This was accomplished at a meeting of the House in which most of the members were absent, and over which Randolph was presiding in the absence of the Speaker.
Randolph resigned as attorney general in 1766. As friction between Britain and the colonies progressed, especially after the House of Burgesses, of which Randolph was by this time Speaker, was dissolved in response to its actions against the Townshend Act, he became more in favor of independence. He chaired meetings of a group of former House members at a Williamsburg tavern, which worked toward responses to the unwelcome tax measures imposed by the British government.
Randolph was selected to chair in both the First and Second Continental Congresses, in large part due to his reputation for leadership while in the House of Burgesses. He did not, however, live to see independence for the nation he led; Randolph died in Philadelphia, and was buried at Christ's Church. He was later re-interred at the College of William and Mary chapel. His nephew, Edmund Randolph, became the first United States Attorney General.