Maggie Mitchell was born in Richmond, Virginia, to former slaves; her father was murdered when she was a child. In 1886, Maggie married Armstead Walker, Jr., a building contractor; they had three children (two of whom survived). She worked first as a teacher, and then as an agent for the Woman's Union Insurance Company. Maggie quickly rose to become the executive secretary/treasurer of the company (which was then called the Independent Order of St. Luke). She also founded a newspaper called the St. Luke Herald (in 1902).

In 1903, Maggie Lena Walker started the St. Luke Penny Savings Bank and was its president. In 1929, at the start of the economic depression, her bank bought all the local black-owned banks in town and renamed itself the Consolidated Bank and Trust Company.

Throughout her life, Walker worked for civil rights and other humanitarian causes, including co-founding the Richmond branch of the NAACP (National Association for the Advancement of Colored People) and supporting the Virginia Industrial School for Colored Girls. Walker's house, at 110 1/2 East Leigh Street, Richmond, Virginia, is designated as an official Historic Site.