Wythe's brother later sent him to Prince George County to read law under an uncle. In 1746, at age 20, he joined the bar, moved to Spotsylvania County, and became associated with a lawyer there. In 1747 he married his partner's sister, Ann Lewis, but she died the next year. In 1754 Lt. Gov. Robert Dinwiddie appointed him as acting colonial attorney general, a position that he held for only a few months. The next year, Wythe's brother died and he inherited the family estate. He chose, however, to live in Williamsburg in the house that his new father-in-law, an architect, designed and built for him and his wife, Elizabeth Taliaferro. They married in 1755, and their only child died in infancy.

At Williamsburg, Wythe immersed himself in further study of the classics and the law and achieved accreditation by the colonial supreme court. He served in the House of Burgesses from the mid-1750s until 1775, first as delegate and after 1769 as clerk. In 1768 he became mayor of Williamsburg, and the next year he sat on the board of visitors of the College of William and Mary. During these years he also directed the legal studies of young scholars, notably Thomas Jefferson. Wythe and Jefferson maintained a lifelong friendship, first as mentor and pupil and later as political allies.

Wythe first exhibited revolutionary leanings in 1764 when Parliament hinted to the colonies that it might impose a stamp tax. By then an experienced legislator, he drafted for the House of Burgesses a remonstrance to Parliament so strident that his fellow delegates modified it before adoption. Wythe was one of the first to express the concept of separate nationhood for the colonies within the British empire.

When war broke out, Wythe volunteered for the army but was sent to the Continental Congress. Although present from 1775 through 1776, Wythe exerted little influence and signed the Declaration of Independence after the formal signing in August 1776. That same year, Wythe, Jefferson, and Edmund Pendleton undertook a 3-year project to revise Virginia's legal code. In 1777 Wythe also presided as speaker of the Virginia House of Delegates.

An appointment as one of the three judges of the newly created Virginia high court of chancery followed in 1778. For 28 years, during 13 of which he was the only chancellor, Wythe charted the course of Virginia jurisprudence. In addition, he was an ex officio member of the state superior court.

Wythe's real love was teaching. In 1779 Jefferson and other officials of the College of William and Mary created the first chair of law in a U.S. institution of higher learning and appointed Wythe to fill it. In that position, he educated America's earliest college-trained lawyers, among them John Marshall and James Monroe. In 1787 he attended the Constitutional Convention but played an insignificant role. He left the proceedings early and did not sign the Constitution. The following year, however, he was one of the Federalist leaders at the Virginia ratifying convention. There he presided over the Committee of the Whole and offered the resolution for ratification.

In 1791, the year after Wythe resigned his professorship, his chancery duties caused him to move to Richmond, the state capital. He was reluctant to give up his teaching, however, and opened a private law school. One of his last and most promising pupils was young Henry Clay.

In 1806, in his eightieth year, Wythe died at Richmond under mysterious circumstances, probably of poison administered by his grandnephew and heir, George Wythe Sweeney. Reflecting a lifelong aversion to slavery, Wythe emancipated his slaves in his will. His grave is in the yard of St. John's Episcopal Church in Richmond.