Cooper enlisted in the army as a private at Louisville, Kentucky in 1861 at the outbreak of the Civil War. He was made a Sergeant on November 15, 1861 and was commissioned as second lieutenant in the 20th Kentucky Infantry on January 24, 1862. From February to July 1862 he was the Acting aide-de-camp (ADC) and Acting Assistant Adjutant general (AAG) for the 22nd Brigade of the 4th Division in the Army of the Ohio. At the Battle of Corinth he said to have "exhibited bravery even to rashness" in an official report by Colonel Thomas D. Sedgewick. In a report that Sedgewick made earlier the same month, he said of Cooper: "Asst. Adjt. Gen. Wickliffe Cooper, as on all occasions before, exhibited the greatest bravery. The coolness and precision with which he made the several reconnaissances ordered by me amid the greatest danger merit the highest praise." In July 1862 he was made the ADC to Maj. General William Nelson. At the Battle of Richmond (August 1862), he was singled out for honors by a Union Army general. He was taken prisoner and paroled on August 30, 1862 in Richmond, Kentucky, as part of a prisoner exchange.