He was the eldest son of Sir George Barne (1532–1592), of Woolwich, and his wife Anne, daughter of Sir William Garrard. His father, a London merchant, had been Lord Mayor of London and an MP for the City of London.
As his father's heir in 1592, he became a Kent landowner and was appointed a Justice of the Peace for the county from 1596. Before then, he was elected to the 1593 Parliament as MP for Great Grimsby. He was knighted in 1603.
Apart from his one foray into national politics and some work for the government, he seems to have spent his life participating in county affairs and managing his lands in Woolwich, Plumstead and Bexley. He also developed literary friendships, among his correspondents being John Chamberlain, Sir Dudley Carleton and Sir Henry Savile.
In 1586, when he was about 24, he married Anne, daughter of Edwin Sandys, the Archbishop of York, and his wife Cecily, daughter of Sir Thomas Wilford and half-sister of Sir James Wilford. Their children included:
- Sir William (born 1593), his heir, who married Dorothy, daughter of the MP Sir Peter Manwood.
- Robert (died after 1632), of Grimsby, who married Elizabeth, daughter of Thomas Twysden and niece of Sir William Twysden, 1st Baronet.
- Miles (born 1600), who became a parson in Kent and married Jane, daughter of Henry Travers, a London lawyer.
- Anne (died 1633), who married Sir William Lovelace, becoming the mother of the poet Richard Lovelace and of Francis Lovelace, Governor of New York.
- W.J.J. (1981), "Barne, William (c.1558-1619), of Tower Place, Woolwich, Kent", in P.W. Hasler (ed.), The History of Parliament: the House of Commons 1558-1603, retrieved 24 January 2018
- Crisp, Frederick Arthur (1893), "Pedigree of Barne", Visitation of England and Wales, retrieved 24 January 2018
- PROB 11/149/9 Will of Edward Pulter of Bradfield, Hertfordshire, 2 May 1626, retrieved 24 January 2018