Killen is the third of four sons and nine children of John Killen (1768–1828), a grocer and seedsman in Ballymena, by his wife Martha, daughter of Jesse Dool, a farmer in Duneane. His paternal grandfather, a farmer at Carnmoney, marries Blanche Brice, a descendant of Edward Brice. A brother, James Miller Killen (1815–1879), is a minister in Comber, County Down. Thomas Young Killen is his father’s great-nephew.
After attending local primary schools, Killen goes to Ballymena Academy around 1816, and in November 1821 enters the collegiate department of the Royal Belfast Academical Institution, under James Thomson. In 1827, he is licensed to preach by the Presbytery of Ballymena, and on November 11, 1829 he is ordained minister at Raphoe, County Donegal.
In July 1841 Killen is appointed, by the General Assembly of the Presbyterian Church in Ireland, its professor of church history, ecclesiastical government, and pastoral theology, in succession to James Seaton Reid. He concentrates on history. When Assembly’s College, Belfast is set up in 1853, he becomes one of the professors there. In 1869 he is appointed president of the college, in succession to Henry Cooke, and undertakes fundraising for professorial endowments and new buildings. In 1889 he resigns his chair but continues as president.
During his career Killen receives the degrees of D.D. (1845) and of LL.D. (1901) from the University of Glasgow. His portrait, painted by Richard Hooke, hangs in the Gamble library of the college.
William Dool Killen dies on January 10, 1902, and is buried in Balmoral Cemetery, Belfast, where a monument marks his resting place.