John Thornley was born August 27, 1816, in King George County, Virginia, to William Thornley and Jane Riding. His father was a planter who owned at least 55 slaves in Caroline County.

John enrolled at the University of Virginia in 1835 and spent the next year studying medicine, chemistry, anatomy, and surgery. He left UVA in 1836 and returned to Caroline County. He enlisted in the United States Navy and passed his examination in July 1839. The following year, on October 19, 1840, the navy appointed him an assistant surgeon.

Thornley served in the Mexican-American War, serving aboard the USS Spitfire and the USS Mississippi. He participated in the Siege of Veracruz in March 1847. In 1848, Thornley traveled to the Holy Land on an expedition assigned to map the Jordan River and the Dead Sea. He was promoted to full surgeon on September 1, 1855. He married Mary Down Pearce in April 1856, and they had one child together: Clarence, born around 1857. Mary died on April 22, 1861, in Morristown, New Jersey, and was buried in Green-Wood Cemetery in Brooklyn, New York.

In May 1861, the naval hospital examined Thornley and declared him “permanently incapable of further service at sea” due to a “highly excitable nervous system.” On June 1, the secretary of the navy ordered him to be placed on the retired list. Thornley, however, secured an agreement to continue serving in the navy while under retired status, and he later sued the federal government to secure longevity pay for his extended term of service. The case went all the way to the Supreme Court, but they dismissed the case.

Thornley remained in the U.S. Navy throughout the Civil War. After the war, he settled in Albemarle County, Virginia, and by 1870 his estate was valued at $60,000. He married Julia Henrietta Payne sometime in the 1860s, and they had at least four children together: Josiah, born around 1867; Julian, born around 1869; Jane, born September 2, 1870; and John, born around 1877. On July 22, 1871, his eldest son Clarence accidentally shot and killed himself at the family home in Charlottesville.

On March 3, 1871, the Navy promoted Thornley to medical director. He retired from the navy for a final time on March 5, 1872. His health, however, remained precarious, and on August 7, 1875, he was admitted to the Naval Hospital in Brooklyn, New York and diagnosed with nervous irritation. In 1887, Thornley briefly secured an $8-per-month pension. He died later that year, on November 9, 1887, in Charlottesville, Virginia. He was buried in Brooklyn with his first wife. After his death, his family received a pension to help support his son John, who was still a minor. His daughter Jane applied for a pension in 1914, but she was ultimately unsuccessful.