Roy was born to Irish-Canadian Elias Charles Disney and English-German-American Flora Call Disney in Chicago, Illinois. The family moved to Marceline, Missouri and to Kansas City in 1911. On July 1, 1911, Elias purchased a newspaper delivery route for The Kansas City Star. It extended from 27th Street to the 31st Street, and from Prospect Avenue to Indiana Avenue. Roy and his brother, Walt worked as newspaper delivery-boys. The family delivered the morning newspaper, The Kansas City Times, to approximately 700 customers, and The Kansas City Star to more than 600. The number of customers served increased with time.

Roy graduated from the Manual Training High School of Kansas City in 1912. He left the paper delivery route and worked on a farm in the summer. He was then employed as a bank clerk along with brother Raymond Arnold Disney at the First National Bank of Kansas City.

Roy served in the United States Navy from 1917 to 1919. Roy contracted tuberculosis and was therefore discharged from military duty. He relocated to Los Angeles and worked as a banker while recuperating in hospital. In 1923, brother Walt joined Roy in Hollywood and the two planned the start of Disney Brothers Studio. The brothers ordered kit houses from Los Angeles-based Pacific Ready Cut Homes, and in 1928, built their homes adjacently on Lyric Avenue in the Los Feliz neighborhood.

While Walt led the creative side, Roy guided the business side and finances. Together Roy and Walt founded Disney Studios as brothers, but Walt later bought out most of Roy’s share in 1929 so, unlike Max and Dave Fleischer of rival Fleischer Studios, Roy was not a co-producer. However, Roy was an equal partner in all facets of the production company.

Roy became the company’s first CEO in 1929, although the official title was not given to him until 1966. He also shared the role of chairman of the board with Walt from 1945. In 1960, Walt dropped the chairman title so he could focus more on the creative aspects of the company. After Walt’s death in 1966, Roy postponed his retirement to oversee construction of what was then known as Disney World. He later renamed it Walt Disney World as a tribute to his brother. Roy became the president of Walt Disney Productions from December 15, 1966, until 1968.

Roy was married to Edna Francis from April 1925 until his death. Their son, Roy Edward Disney, was born on January 10, 1930. Throughout his life, Roy rejected the publicity and fame that came with being Walt’s brother. Roy’s nephew Charles Elias Disney chose to name his son Charles Roy Disney in Roy’s honor.

After the opening of Walt Disney World in October 1971, Roy finally retired. He died, aged 78, on December 20, 1971, from an intracranial hemorrhage. He is interred in Forest Lawn Memorial Park (Hollywood Hills) next to his wife Edna Francis Disney in Los Angeles.

One of the Walt Disney World Railroad locomotives was named after Roy. On June 6, 2002, his son Roy E. Disney rededicated this locomotive in his father’s honor. As of 2016, this locomotive turned a hundred years old. One of the Hong Kong Disneyland Railroad locomotives is also named the Roy O. Disney.

In 2014, Roy O. Disney was portrayed in the feature film Walt Before Mickey by Jon Heder.