Blair was born in 1741 in Faggs Manor, near Cochranville, Chester County, Pennsylvania, the son of a Presbyterian minister also named Samuel Blair. He graduated from The College of New Jersey in 1760 and was licensed to preach in 1764. A conscientious and eloquent minister, he became pastor of the Old South Church in Boston in 1766. In 1767, at the age of 26, he was offered the presidency of the College of New Jersey (now Princeton University) but voluntarily stepped aside when John Witherspoon (later a signatory of the Declaration of Independence) became available.
Blair remained at the Old South Church until 1769, when he resigned his charge because of ill health. He then made his residence at Germantown, Pennsylvania, where he devoted the rest of his life to study. In 1790 he earned the degree of Doctor of Divinity from the University of Pennsylvania, and was appointed Chaplain in Congress on December 10, 1790, a position he held for two years until the election of Ashbel Green on November 5, 1792. Blair died in September 1818.