Mary Walker was born in the Town of Oswego, New York, in 1832, the daughter of Alvah (father) and Vesta (mother) Walker. She was the youngest of five daughters and had one younger brother. Walker worked on her family farm as a child. She did not wear women's clothing during farm labor, because she considered them too restricting. Her elementary education consisted of going to the local school where her mother taught. As a young woman, she taught at the school to earn enough money to pay her way through Syracuse Medical College, where she graduated as a medical doctor in 1855 as the only woman in her class. She married a fellow medical school student, Albert Miller, and they set up a joint practice in Rome, New York. The practice did not flourish, as female physicians were generally not trusted or respected at that time.

At the beginning of the American Civil War, she volunteered for the Union Army as a civilian. At first, she was only allowed to practice as a nurse, as the Army had no female surgeons. During this period, she served at the First Battle of Bull Run  July 21, 1861 and at the Patent Office Hospital in Washington, D.C. She also worked as an unpaid field surgeon near the Union front lines, including the Battle of Fredericksburg and in Chattanooga after the Battle of Chickamauga. Finally, she was awarded a commission as a "Contract Acting Assistant Surgeon (civilian)" by the Army of the Cumberland in September, 1863, becoming the first-ever female U.S. Army Surgeon. (Manassas), 

Walker was later appointed assistant surgeon of the 52nd Ohio Infantry. During this service, she frequently crossed battle lines, treating civilians. On April 10, 1864, she was captured by Confederate troops and arrested as a spy. She was sent to Richmond and remained there until August 12, 1864 when she was released as part of a prisoner exchange. She went on to serve during the Battle of Atlanta and later as supervisor of a female prison in Louisville, Kentucky, and head of an orphanage in Tennessee.

Walker was recommended for the Medal of Honor  by generals   William Tecumseh Sherman and George Henry Thomas.  On November 11, 1865, President Andrew Johnson signed a bill to present her the medal, specifically for her services at the First Battle of Bull Run (Manassas).

In 1917, the U.S. Congress, after revising the standards for award of the medal so that it could only be given to those who had been involved in "actual combat with an enemy", revoked more than 900 previously-awarded medals, including that of Dr. Mary Edwards Walker and William F. "Buffalo Bill" Cody. 

Although ordered to return the medal, she refused to do so and continued to wear it until her death.

President Jimmy Carter restored her medal posthumously in 1977.

In World War II, a Liberty ship, the SS Mary Walker, was named for her.

In 1982, the U.S. Postal Service issued a 20 cent stamp in her honor.

The medical facilities at SUNY Oswego are named in her honor. On the same grounds a plaque explains her importance in the Oswego community.

There is a United States Army Reserve center named for her in Walker, Michigan.

The Whitman-Walker Clinic in Washington, D.C. is named in honor of Dr. Walker and the poet Walt Whitman who was a nurse in D.C. during the Civil War.