Raised in Washington, DC, Southworth studied in a school kept by her stepfather, Joshua L. Henshaw, and in 1840 married inventor Frederick H. Southworth, of Utica, New York. E.D.E.N. Southworth moved with her husband out to Wisconsin to become a teacher. After 1843, she returned to Washington, D.C. without her husband and with two young children.
She began to write stories to support herself and her children when her husband deserted her in 1844. Her first story, "The Irish Refugee", was published in the Baltimore Saturday Visitor. Some of her earliest works appeared in The National Era, the newspaper that printed Uncle Tom's Cabin. The bulk of her work appeared as a serial in Robert Bonner's New York Ledger,and in 1857 Southworth signed a contract to write exclusively for this publication.
Like her friend Harriet Beecher Stowe, she was a supporter of social change and women's rights, but she was not nearly as active on these issues. Her first novel, Retribution, a serial for the National Era, published in book form in 1846, was so well received that she gave up teaching and became a regular contributor to various periodicals, especially the New York Ledger. She lived in Georgetown, D.C., until 1876, then in Yonkers, New York, and again in Georgetown, D.C., where she died.