McCook was born in New Lisbon, Ohio, to Dr. John McCook. He learned the printing trade as a youth, then taught school for several years. attended Jefferson College. After graduation in 1859, he studied theology privately and in the Western Theological Seminary in Allegheny, Pennsylvania. With the outbreak of the Civil War, he enlisted as a chaplain in the 41st Illinois Infantry as a lieutenant, and helped tend the wounded. As a minister in Clinton, Illinois, St. Louis, and Steubenville, Ohio, McCook became known for his compassion and intellect, and for his leadership in the movement to create Sunday Schools. In 1869, he became pastor of the Seventh Presbyterian church of Philadelphia, where he lived for the rest of his life.
He spent his summers studying the behavior of ants and spiders. He published his observations and discoveries in a number of journals and books, as well as in a series of well-received illustrated children's books that explained the insects characteristics and traits in language and drawings for young minds. Many of McCook's books used illustrations drawn by Daniel Carter Beard, the founder of the Boy Scouts of America.
In the summer of 1877, he travelled to Texas to study agricultural ants. Two years later, McCook wrote The Natural History of the Agricultural Ant of Texas. In 1889–93, he published his most ambitious work, American Spiders and Their Spinning Work, in three illustrated volumes. He also wrote a book on his ancestors in the Whiskey Rebellion, and delivered a number of papers on Civil War history at meetings of the Military Order of the Loyal Legion of the United States veterans organization.
McCook was Vice President of both the American Entomological Society and the Academy of Natural Sciences. In 1880, Lafayette College conferred the degree of Doctor of Divinity to McCook. In 1895, he designed the official flag of the city of Philadelphia. He again served as an Army Chaplain during the Spanish-American War in 1898.