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Margaret Long was born in 1873 to Mary Woodward Glover and John Davis Long, a politically ambitious lawyer who practiced in Hingham, Massachusetts. John Long was elected to the Massachusetts General Court in 1874 and from there embarked on a career with the Republican Party, serving as governor of Massachusetts from 1880 to 1883 and secretary of the Navy in President William McKinley's cabinet from 1897 to 1902. Mary Glover Long died in 1882; John Long remarried Agnes Peirce in 1886 and had a son, Peirce, in addition to Margaret and her sister, Helen, from his first marriage. The family lived in Hingham, Massachusetts and owned a vacation property in Buckfield, Maine.

Margaret Long entered Smith College in 1891. Among her fellow students were Florence Rena Sabin, class of 1893, and Dorothy Reed (Mendenhall), class of 1895. At Smith, she and Reed lived in Wallace House and belonged to a tongue-in-cheek social club, the "Anti-Matrimonial Alliance." After Long's graduation in 1895, she traveled and studied in Europe, undertook preparation for medical school at the Massachusetts Institute of Technology, and entered the Johns Hopkins Medical School three years behind Reed. Reed, Sabin, and Long came to distinguish themselves as graduates of the new medical school and pioneer researchers and activists in medicine and public health.

Long was awarded a M.D. in 1903, and in 1905, she moved to Colorado to recover from tuberculosis, from which her sister had died in 1901. Anticipating the public health work of Florence Sabin, who left a position on the Johns Hopkins faculty to implement a public health program in Colorado in the 1940s, Long became active in the fight against tuberculosis. In addition to her epidemiological research in Denver area hospitals, she helped found the Sands House Sanatorium and served on its board for many years.

After her retirement in the 1940s, Long took an interest in the history of the West. She had a passion for the trails of the pioneers and published her reconstructions and research of their paths in several travel-oriented works, including The Shadows of the Arrow (1941), The Smoky Hill Trail (1953), The Oregon Trail (1954), and The Sante Fe Trail (1954). She also published a book of poetry, The Enchanted Desert (1942), and edited volumes of family papers. Her edition of her father's journal, published in 1956, remains a significant source for research into one of the primary administrators of the Navy during the Spanish-American War and a party to Theodore Roosevelt's controversial and imperialistic tenure in the Department of the Navy.

Margaret Long's later years were spent among a community of bachelor women in Denver, which included her long-time companion, Elsie Pratt, and sisters Florence and Mary Sabin. Long died in 1957.