Singleton was born in Los Angeles, the son of Sheila Ward-Johnson, a pharmaceutical company sales executive, and Danny Singleton, a real estate agent, mortgage broker, and financial planner. He attended Blair High School, Pasadena City College and the USC School of Cinematic Arts. He graduated from USC in 1990, and is a member of Kappa Alpha Psi fraternity. Singleton was enrolled in the University of Southern California's Filmic Writing program under Margaret Mehring and her now-famous curriculum. The program was designed to take students directly into the Hollywood system as proficient writer/directors.

Singleton's 1991 film debut Boyz n the Hood, an inner city drama starring Cuba Gooding, Jr., Ice Cube, and Laurence Fishburne, was both a significant critical and commercial success. For his efforts, Singleton received Academy Award nominations for Best Original Screenplay and Best Director. At age 24 he was the youngest person ever nominated for Best Director, and the first African-American to be nominated for the award (four others, Lee Daniels, Steve McQueen, Barry Jenkins and Jordan Peele, have been nominated since). The film has since attained classic status and, in 2002, the United States Library of Congress deemed the film "culturally significant" and selected it for preservation in the National Film Registry.

He has four children. John has a daughter named Justice Maya Singleton (born on October 17, 1992) with former wife Tosha Lewis, as well as a son, Maasai Mohandas Singleton (born April 3, 1994).

Singleton's follow-up films Poetic Justice (1993) and Higher Learning (1995) were similarly socially conscious and received mixed to positive reviews. Singleton also directed the video for Michael Jackson's 1991 single "Remember The Time", which featured Eddie Murphy, Iman and Magic Johnson.

On October 12, 1996, John married Ghanaian Actress Akosua Gyamama Busia, who is the daughter of Ghana's second Prime Minister Dr. Kofi Abrefa Busia. They have a daughter named Hadar Singleton, born on April 3, 1997, who appeared in Tears of the Sun (2003) and other films. Singleton and Busia divorced in June 1997. Singleton also has a daughter, Cleopatra Singleton, who was born in 1998.

In 1997, his film Rosewood, a historical drama about racial violence, was entered into the 47th Berlin International Film Festival. Both this and his 2001 film Baby Boy received very positive reviews and helped establish Singleton's critical reputation. Additionally, his adaptation of Shaft (2000), starring Samuel L. Jackson in the title role, was successful critically and commercially.

Singleton has since turned to directing action films, such as 2 Fast 2 Furious (2003) and Four Brothers (2005), which have for the most part received mixed to negative reviews.

In 2005, Singleton teamed with Craig Brewer and financed the independent film Hustle and Flow, once it was clear that most other major backers would not clear it for release.

On August 23, 2007, Singleton was involved in an automobile accident in which he struck a jaywalking pedestrian, Constance Russell, 57, of Los Angeles. Staying on the scene until police arrived, Singleton was not under the influence of alcohol or other substances, and was released after being questioned. Russell died later in the hospital. The case was turned over to the District Attorney but no charges were filed.

On March 19, 2014, Singleton criticized popular studios for "refusing to let African-Americans direct black-themed films". Singleton told an audience of students at Loyola Marymount University "They ain't letting the black people tell the stories." He also added, "They want black people [to be] what they want them to be. And nobody is man enough to go and say that. They want black people to be who they want them to be, as opposed to what they are. The black films now — so-called black films now — they're great. They're great films. But they're just product. They're not moving the bar forward creatively. … When you try to make it homogenized, when you try to make it appeal to everybody, then you don't have anything that's special."

In 2013, it was announced that Singleton was attached as Director of the Tupac Shakur biography film. On April 3, 2015, Singleton reported that production was put on hold. Four days later, it was announced that following creative differences with Morgan Creek Productions, Singleton had stepped down as Director, and was replaced by Carl Franklin. Singleton also stated he was planning on making a competing film about Tupac.