Duff's parents had established a commercial outpost in Guling, near Jiujiang, and he was sent back to Hamilton, Ontario, for schooling, remaining there until age 16 before returning to China. When the First World War broke out in 1914, he traveled across Russia to England and joined the army. He was gravely wounded at the Third Battle of Ypres and was sent to a hospital in England, where he met and married his nurse. He learned to drive in 1919 and entered his first race in 1920.
Duff began racing at Brooklands in 1920, initially at the wheel of a 10-litre Fiat S.61 built in 1908. By August of that year he was lapping in the same speed range as Henry Segrave, one of the leading Brooklands and Grand Prix drivers of the era. He won the 75 Long Handicap at Brooklands in May 1921 at 104.19 mph and the 100 Long Handicap at 104.85 mph at the mid-summer meeting. He later drove a 21-litre Blitzen Benz at Brooklands' autumn meeting, lapping at 114.49 mph before losing control at the end of the race and crashing through trees and a telegraph pole beyond the banking.
In 1922 Duff co-founded the dealership Duff and Aldington to sell Bentley cars, which introduced him to the marque he would make famous. He conducted extensive record attempts at Brooklands with a 3-litre Bentley, including a solo Double 12 run on September 27–28 at an average of 86.52 mph covering 2,082 miles and setting 38 international class records.
In early 1923, Duff learned of a new 24-hour race planned for Le Mans and became the first entrant. W. O. Bentley, skeptical the race would be survivable for any car, agreed to prepare Duff's entry and assigned factory test driver Frank Clement as co-driver. The pair set the fastest lap of the 1923 race but a flying stone pierced the fuel tank, forcing Clement to bicycle back to the stricken car with fuel before they could return to the pits. Despite the drama, Duff and Clement finished fourth. Bentley, who attended only at the last moment, became committed to Le Mans as a result.
By 1924 Bentley was fully engaged at Le Mans. Duff remained a private entrant but his car was prepared alongside the factory effort using ideas he had developed after the 1923 race. Racing in intense heat with Clement again as co-driver, Duff won convincingly to give Bentley its first Le Mans victory. In 1925, a carburetor fire ended his Le Mans effort before he could defend the title.
On September 9–10, 1925, Duff traveled to the Montlhéry circuit near Paris with co-driver Dudley Benjafield for an assault on the absolute 24-hour distance record. Driving in heavy rain in a Bentley fitted with a special single-seater Weymann body, they covered the first 12 hours at 97.7 mph before the camshaft drive failed at 18.5 hours. They claimed world records for 1,000 kilometres and 1,000 miles.
Duff returned to Montlhéry on September 21 with Woolf Barnato as co-driver. On a damp, misty track the pair covered 2,280 miles in 24 hours at an average of 95.02 mph, bettering the previous absolute record — held by a 9-litre Renault — by more than 7 mph. The 3-litre Bentley took 21 world records during the run, including records for six and twelve hours and 500, 1,000, and 2,000 miles.
In February 1926 Duff traveled to the United States and signed to drive an Elcar-sponsored Miller in the Indianapolis 500 following the death of the car's previous driver. In a race shortened to 400 miles by rain, he finished ninth. He subsequently raced on the Altoona board track, finishing third in the 250-mile event. At Rockingham in Salem, New Hampshire, a puncture threw his car sideways and he was ejected, suffering a broken collarbone, painful muscle injuries, and a concussion that affected his vision. Having promised his wife he would retire if seriously hurt again, he kept his word and ended his racing career.
After retiring from racing, Duff settled his family in Santa Monica near Los Angeles and opened a fencing academy, training many Hollywood film stars of the era. He doubled for his friend Gary Cooper in swordfighting sequences and taught fencing at UCLA. In 1932 he coached members of the United States Olympic fencing team. Economic pressures during the Depression reduced demand for fencing instruction and the family relocated to China from 1932 to 1934, then returned to England. In January 1935 Duff was admitted to the British Racing Drivers' Club. He continued to compete successfully as a steeplechase and show jumping rider well into the late 1930s. John Duff died following a riding accident in Epping Forest on January 8, 1958.
Duff is a member of the Canadian Motorsport Hall of Fame. His private entry at the 1923 Le Mans — the first ever — was instrumental in convincing W. O. Bentley to commit factory resources to the race, leading directly to the Bentley Boys era that produced five Le Mans victories between 1924 and 1930. Without Duff's initial conviction and persistence, the most celebrated chapter of Bentley's racing history may never have begun.