Her elementary education consisted of attendance at the local school that her parents had started. The Walkers were determined that their daughters be as well-educated as their son, so they founded the first free schoolhouse in Oswego in the late 1830s. After finishing primary school, Mary and two of her older sisters attended Falley Seminary in Fulton, New York. Falley was not only an institution of higher learning, but a place that emphasized modern social reform in gender roles, education, and hygiene. Its ideologies and practices further cemented Mary's determination to defy traditional feminine standards on a principle of injustice. In her free time, Mary would pore over her father's medical texts on anatomy and physiology; her interest in Medicine is attributable to her exposure to medical literature at an early age. As a young woman, she taught at a school in Minetto, New York, eventually earning enough money to pay her way through Syracuse Medical College (now the State University of New York Upstate Medical University), where she graduated with honors as a medical Doctor in 1855, the only woman in her class.

Rank and organization: Contract Acting Assistant Surgeon (civilian), U. S. Army. Places and dates: Battle of Bull Run, July 21, 1861; Patent Office Hospital, Washington, D.C., October 1861; Chattanooga, Tenn., following Battle of Chickamauga, September 1863; Prisoner of War, April 10, 1864 – August 12, 1864, Richmond, Va.; Battle of Atlanta, September 1864. Entered Service at: Louisville, Ky. Born: 26 November 1832, Oswego County, N.Y.

She married a fellow medical school student, Albert Miller, on November 16, 1855, shortly before she turned 23. Walker wore a short skirt with trousers underneath, refused to include "obey" in her vows, and retained her last name, all characteristic of her obstinate nonconformity. 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. They later divorced, on account of Miller's infidelity.

Walker briefly attended Bowen Collegiate Institute (later named Lenox College) in Hopkinton, Iowa, in 1860, until she was suspended for refusing to resign from the school's debating society, which until she joined had been all male.

Despite having kept a private practice for many years, Walker was compelled at the outbreak of American Civil War to volunteer as a surgeon - first for the Army but was rejected because she was a woman. She was offered the role of a nurse but declined and chose to volunteer as a surgeon for the Union Army. as a civilian. The U.S. Army had no female Surgeons, and at first, she was allowed to practice only as a nurse. During this period, she served at the First Battle of Bull Run (Manassas), July 21, 1861, and at the Patent Office Hospital in Washington, D.C. She worked as an unpaid field surgeon near the Union front lines, including at the Battle of Fredericksburg and in Chattanooga after the Battle of Chickamauga. As a suffragist, she was happy to see women serving as Soldiers, and alerted the press to the case of Frances Hook, in Ward 2 of the Chattanooga hospital, a woman who served in the Union forces disguised as a man. Walker was the first female surgeon of the Union army. She wore men's clothing during her work, claiming it to be easier for high demands of her work.

In September 1862, Walker wrote to the War Department requesting employment as a spy, but her proposal was declined. In September 1863, she was employed as a "Contract Acting Assistant Surgeon (civilian)" by the Army of the Cumberland, becoming the first female surgeon employed by the U.S. Army Surgeon. Walker was later appointed assistant surgeon of the 52nd Ohio Infantry. During her Service, she frequently crossed battle lines and treated civilians.

On April 10, 1864, she was captured by Confederate troops, and arrested as a spy, just after she finished helping a Confederate Doctor perform an amputation. She was sent to Castle Thunder in Richmond, Virginia, and remained there until August 12, 1864, when she was released as part of a prisoner exchange. While she was imprisoned, she refused to wear the clothes provided her, said to be more "becoming of her sex". Walker was exchanged for a Confederate surgeon from Tennessee on August 12, 1864.

After the war, 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 award her the medal.

In 1870, Dr. Walker was arrested in New Orleans and mocked by men because she was dressed as a man. The arresting officer Mullahy twisted her arm and asked her if she had ever had sex with a man. Dr. Walker was released from custody when she was recognized at Police Court. New Orleans Republican February 22, 23, 1870. This incident was also cited by Perry Young in the Mistick Krewe, Chronicles of Comus and His Kin, 1931, 159.

Walker was a member of the central woman's suffrage Bureau in Washington, and solicited funds to endow a chair for a woman professor at Howard University medical school. She attempted to register to vote in 1871, but was turned away. The initial stance of the movement, following her lead, was to claim that women already had the right to vote, and Congress needed only to enact enabling legislation. After a number of fruitless years advocating this position, the movement promoted the adoption of a constitutional amendment. This was diametrically opposed to her position, and she fell out of favor with the movement. She continued to attend suffrage conventions and distribute her own literature, but was virtually ignored by the rest of the movement. Her penchant for wearing masculine clothing, including a top hat, only exacerbated the situation. She received a more favorable reception in England than in the United States.

In 1907, Walker published "Crowning Constitutional Argument", in which she argued that some States, as well as the federal Constitution, had already granted women the right to vote. She testified on women's suffrage before committees of the U.S. House of Representatives in 1912 and 1914.

In 1917, the U.S. Congress created a pension act for Medal of Honor recipients, and in doing so created separate Army and Navy Medal of Honor Rolls. Only the Army decided to review eligibility for inclusion on the Army Medal of Honor Roll. The 1917 Medal of Honor Board deleted 911 names from the Army Medal of Honor Roll, including those of Dr. Mary Edwards Walker and william F. "Buffalo Bill" Cody. The disenrolled recipients were ordered to return their medals, but Walker continued to wear hers until her death.

After a long illness, Walker died at home on February 21, 1919, at the age of eighty-six. She was buried at Rural Cemetery in Oswego, New York, in a plain funeral, with an American flag draped over her casket, and wearing a black suit instead of a dress. Her death in 1919 came one year before the passage of the Nineteenth Amendment to the United States Constitution, which guaranteed women the right to vote.

President Jimmy Carter restored her medal posthumously in 1977. She is one of six people to regain the award.

In 1982, the U.S. Postal Service issued a twenty-cent stamp in her honor, marking the anniversary of her birth.

Walker was inducted into the National Women's Hall of Fame in 2000.

In May 2012, a 900-pound bronze statue honoring Walker was unveiled in front of the Oswego, New York Town Hall.