Douglas Day was born on April 27, 1829, in Virginia, to Baldwin Day and Lucretia Guthrie. His father was a wealthy merchant and a veteran of the War of 1812. The family lived in Fauquier County, Virginia, where Baldwin owned at least 13 slaves. Douglas enrolled at the University of Virginia in 1850, studying chemistry, anatomy, medicine, and surgery. He earned his medical degree on June 27, 1851. After graduation, he spent a year at the University of Pennsylvania, where he specialized in the structure of the circulatory system.

Day moved to Zanesville, Ohio, by 1854, and on October 1, 1857, he married Martha Virginia Turner in Muskingum County. They had five children: Anna, born 1859; Baldwin, born 1861; Alexandra, born 1865; Virginia, born 1868; and Douglas, born 1874. By 1860, they were living in Zanesville, where Day worked as a physician. On May 15, 1861, he enlisted as a surgeon in the 22nd Ohio Infantry, a 3-month regiment. They left Ohio two weeks later, contending with Confederate guerrillas in present-day West Virginia. He resigned in early July and returned to Zanesville, where he resumed his medical practice.

By 1868, the family had moved back to Fauquier County, Virginia, where they lived with his widowed mother. Day died on January 27, 1875, in Warrenton, Virginia.