Knowles was raised in Gadsden, Alabama, the son of Lue Helen and Matthew Q. Knowles (April 4, 1927 – December 30, 1996). He is a 1974 graduate of Fisk University in Nashville, Tennessee, where he earned both a Bachelor of Arts degree in Economics and a Bachelor of Science degree in Business Administration. Additionally Knowles has an MBA from Cornerstone Christian Bible College for Strategic Leadership and Organizational Culture. Knowles is a visiting professor at Texas Southern University, where he teaches courses on the entertainment industry in the School of Communications. Knowles is the recipient of the Century Award of Excellence (1911–2011) from the Omega Psi Phi fraternity. He also received the 2011 Living Legends Foundation Award and was named the "2007 International Executive of the Year" by the Greater Houston Partnership. Knowles is an active and longtime voting member of the National Academy of Recording Arts and Sciences (NARAS) and serves on its GRAMMY Board Committee. In 2011, he was appointed to the Board of Directors of the Gospel Music Association.

Knowles began his Business career in sales of office and medical equipment. He moved to Houston in 1976 and worked for the Xerox Corporation for 10 years, primarily in the medical-systems sales division. Knowles afterward worked selling a CT/MRI diagnostic imaging equipment to hospitals and health facilities. He also worked in neurosurgical sales. In 1992, Knowles left this career to devote himself to his music company, Music World Entertainment, which he founded that year.

In 2002 Knowles founded Spirit Rising Music, which became Music World Gospel. Its artists include Vanessa Bell Armstrong, Brian Courtney Wilson, Micah Stampley, Juanita Bynum, Le'Andria Johnson, Amber Bullock, Andrea Helms, Trinitee 5:7, and Elder Goldwire. In 2016, through Music World Music, Knowles acquired the country music catalog Compadre, which includes Johnny Cash, Billy Joe Shaver, James McMurty, Trent Willmon, and Kate Campbell. Knowles also launched the family-oriented music imprint, Music World Kids. The label has released youth projects that include Baby Jamz, Krazy Kuzins, Kid's Rap Radio, and the Music World Kids CD 5-pack. The venture also has released music from Nickelodeon's popular cartoon series, Wow! Wow! Wubbzy!.

In addition to his work in music, Knowles is also one of the executive producers of the 2009 movie Obsessed, which stars Beyoncé. He is also executive Producer of, and is featured in, two reality series: The UK's Chancers, and MTV International's Breaking From Above.

In 2013, Knowles was accused of not paying taxes for the years 2010 and 2011. The total amount Knowles owed was reported to be $1,213,580.84.

In October 2015, Mathew Knowles released his first book, "The DNA Of Achievers: 10 Traits of Highly Successful Professionals". In an interview with Madamenoire.com, he spoke about teaching entrepreneurship on the college level, and his entertainment industry seminars.