He was born of an old Virginia family in Stafford County, Virginia. His father was a wealthy gentleman farmer, a slaveholder, and county judge whose home still stands in Falmouth, Virginia along the Rappahannock River. Conway's mother was a homemaker and homeopathic physician. Both parents were Methodists, his father having left the Episcopal church, his mother the Presbyterian. Moncure's opposition to slavery came from his mother and from his boyhood experiences. His father and three brothers remained staunchly pro-slavery.

He graduated at Dickinson College in 1849, studied law for a year, and then became a Methodist minister in his native state. In 1852, thanks largely to the influence of Ralph Waldo Emerson, his religious and political views underwent a radical change, and he entered the Harvard University school of divinity, where he graduated in 1854. Here he fell under the influence of "transcendentalism," and became an outspoken abolitionist. After graduation from Harvard University, Conway accepted a call to the First Unitarian Church of Washington, D.C., where he was ordained in 1855, but his anti-slavery views brought about his dismissal in 1856.

On his return to Virginia, his abolitionist stance and his rumoured connection with the attempt to rescue the fugitive slave, Anthony Burns, in Boston, Massachusetts, aroused the bitter hostility of his old neighbours and friends. In consequence, he left the state. From 1856 to 1861 he was a Unitarian minister in Cincinnati, Ohio, where, also, he edited a short-lived liberal periodical called The Dial.

While in Cincinnati, Conway married Ellen Davis Dana. Ellen was a member of the Unitarian faith, a feminist and an abolitionist. The couple had four children. Despite the previous tension with his family over slavery issues, Conway nevertheless brought his bride to meet his family. His wife broke a Southern social constraint by hugging and kissing a young slave girl in front of family members. After this, it would take seventeen years before Conway reconciled with his family.

Subsequently he became editor of the Commonwealth in Boston, and wrote The Rejected Stone (1861) and The Golden Hour (1862), both powerful pleas for emancipation. In 1862, after spending more and more time away from his church advancing the abolitionist cause, Conway left its ministry. He had grown dissatisfied with the theological, liturgical, and social conservatism of mainstream Unitarianism. After that, he maintained an uneasy and uncertain relationship with Unitarianism, in America and subsequently in England, until he made a clean break.

In 1863, Conway was asked by American abolitionists to go to London to convince the United Kingdom that the American Civil War was a war of abolition. Under English influence, Conway eventually contacted the Confederate States of America "on behalf of the leading antislavery men of America," offering the preservation of the Confederacy after the war's end in exchange for emancipation of the slaves. His support by his sponsors was quickly and angrily withdrawn. Rather than go back to America, where he no longer felt welcome, he went briefly to Venice, Italy, where he was reunited with his wife and children.

Upon return to London, he became the minister of the South Place Chapel, Finsbury, London. The congregation and Conway soon left fellowship with the Unitarian Church. During this time, Conway wrote frequently for the London press. The congregation today is called the South Place Ethical Society.

In 1864, he abandoned theism after one of his sons died. His thinking continued to move from Emersonian transcendentalism toward a more humanistic "freethought". In 1868 Conway was one of four speakers at the first open public meeting in support of women's suffrage in Great Britain. Conway's many literary and intellectual friends included Charles Dickens, Robert Browning, Thomas Carlyle, Charles Lyell, and Charles Darwin.

In the 1870s and 1880s, he returned on and off to the United States. In 1875, he reconciled with his family. In 1897, Conway and his wife returned from London to New York City. Ellen, terminally ill, wished to die in the United States of America. She died on Christmas Day. As the Spanish American War approached, Conway became disaffected with his countrymen. He moved to France to devote much of the rest of his life to the peace movement and writing. Conway died alone in his Paris apartment on November 15, 1907.

In addition to those publications mentioned above, Conway's publications include:

Tracts for To-day (1858)
The Natural History of the Devil (1859)
Testimonies Concerning Slavery (1864)
The Earthward Pilgrimage (1870)
Republican Superstitions (1872)
Idols and Ideals (1871)
Demonology and Devil Lore (2 vols., 1878)
A Necklace of Stories (1879)
Thomas Carlyle (1881)
The Wandering Jew (1881)
Emerson at Home and Abroad (1882)
Pine and Palm (2 vols., 1887)
Life and Papers of Edmund Randolph (1888)
The Life of Thomas Paine with an unpublished sketch of Paine by William Cobbett (2 vols., 1892)
Solomon and Solomonic Literature (1899)
his Autobiography (2 vols, 1900)
My Pilgrimage to the Wise Men of the East (1906).