Turner Ashby, Jr. was born at Rose Bank Plantation in Fauquier County, Virginia, to Turner Sr. and Dorothea Green Ashby. As a child he often played in the waters of nearby Goose Creek. His father died when he was young, and Turner was raised by his mother. In later years, he bought a residence near his childhood home and named it Wolfe's Crag. His father had fought as a colonel in the War of 1812, and his grandfather Jack Ashby served as a captain during the American Revolutionary War.

Ashby was privately educated. Prior to military service he was engaged in business and farming, enjoying modest success at both. He was also known for his chivalry: when a young male guest at a party insulted Ashby and called him out to a duel, Ashby insisted that since he was the host, he would not duel the guest, who being young and inexperienced with guns was unlikely to walk away alive.

An accomplished horseman at an early age, Ashby in his 20s organized a cavalry company of his friends known as the Mountain Rangers. The Mountain Rangers were absorbed into the Virginia Militia in 1859 following John Brown's raid at Harpers Ferry; they performed guard duty at Charles Town during Brown's trial and execution. Ashby made the statement that the Civil War really began with John Brown's insurrection. Ashby was an avid follower of politics and ran for the state legislature, but was a Whig (the minority party in Fauquier County) and follower of Henry Clay, and was not elected. After the start of the Civil War, though he'd disapproved of secession, when it became obvious that Virginia would secede, Ashby persuaded Governor John Letcher to order the militia to capture the federal arsenal at Harpers Ferry. When secession was approved, Ashby made his move, but U.S. forces burned most of the arsenal buildings and 15,000 small arms before he could arrive.

At Harpers Ferry, Ashby was assigned to the Virginia Militia command of Colonel Thomas J. "Stonewall" Jackson. He was responsible for guarding fords across the Potomac River and bridges from Harpers Ferry to Point of Rocks, Maryland. His command assisted Maryland men with Confederate sympathies to pass into Virginia, and they disrupted railroad traffic on the Baltimore and Ohio Railroad and interfered with the passage of boats on the Chesapeake and Ohio Canal. Ashby suffered a personal loss when his brother Richard was killed during an engagement with a Union patrol along the Potomac in June 1861. Ashby, convinced his brother had been bayoneted while trying to surrender after he had a chance to examine his corpse, came to hate Northerners and desired revenge.

On July 23, 1861, Brigadier General Joseph E. Johnston appointed Ashby lieutenant colonel of the 7th Virginia Cavalry. Due to the illness of the regimental commander, Ashby had effective control of half of the regiment, which he operated separately. When the commander retired in February 1862, Ashby assumed command of the entire regiment on March 12. Ashby organized the first Confederate horse artillery, named Chew's Battery, as part of this regiment. The 7th did not participate directly in the First Battle of Manassas, but Ashby aided the Confederate cause by screening the movement of Johnston's army to the Manassas area. The Union had hoped that Johnston's forces would be pinned down by Major General Robert Patterson, but Ashby's screen allowed Johnston to move freely without Patterson's interference.

By the spring of 1862, the 7th Virginia Cavalry had reached the enormous size of 27 infantry and cavalry companies, much larger than a typical Civil War regiment. Stonewall Jackson, in overall command of the Shenandoah Valley, tried to correct the situation by stripping Ashby of his cavalry forces, ordering them to be assigned to two infantry brigades. Ashby threatened to resign in protest and Jackson backed down. Jackson continued to resist Ashby's promotion to brigadier general, due to his informal military training and consequent lack of discipline. Nevertheless, Ashby's promotion came through on May 23, 1862, and he received his promotion and general's star in a ceremony at the Taylor Hotel in Winchester, Virginia.

Ashby cut a striking figure, called by many the "Black Knight of the Confederacy". He generally rode horses that were pure white or pure black. A civilian in the Valley named Thomas A. Ashby (no relation) wrote about an encounter with him:

He was just entering upon a career that soon made him an heroic character in the history of the Civil War. Dressed now in Confederate gray, with gilt lace on his sleeves and collar, wearing high top - boots with spurs and a broad - brimmed black felt hat with a long black feather streaming behind, his appearance was striking and attractive. He stood about five feet eight inches in height and probably weighed from 150 to 160 pounds (68 to 73 kg). He was muscular and wiry, rather thin than robust or rugged. His hair and beard were as black as a raven's wing; his eyes were soft and mahogany brown; a long, sweeping mustache concealed his mouth, and a heavy and long beard completely covered his breast. His complexion was dark in keeping with his other colorings. Altogether, he resembled the pictures I have seen of the early Crusaders, — a type unusual among the many men in the army, a type so distinctive that, once observed, it cannot soon be forgotten.