Johnson was born and raised in San Jose, California, in a devout Christian family and is herself a Christian. She is of Mexican and Native American descent. She was a Pop Warner Cheerleader from the age of 8 and was active in soccer, softball, and track.; she also practiced hip hop and break dancing. She began acting as a senior in high school and was especially interested in imitating different accents. She later studied speech communications at De Anza College. She subsequently became a Cheerleader for the Oakland Raiders after being inducted in 2002. As a member of the Oakland Raiderettes, she was named Rookie of the Year and performed at Super Bowl XXXVII.

Johnson moved to Los Angeles in 2005 to pursue a career in comedy after a friend suggested that she join a joke writing class. She took improv classes, and quickly began to headline her own shows. Her stand-up routine, comprising her impression of a Vietnamese nail salon employee, received a lot of attention on YouTube. In 2007, Johnson joined the cast of Sketch comedy show MADtv as a featured performer. Due to the impending Writers strike, Johnson was often given only a few lines in the scripts during her tenure, as the Writers had to produce more scripts at a faster pace. However, her character Bon Qui Qui, a fast food employee who treats customers rudely, which she wrote herself, was received well, and gained a similarly large YouTube following. In 2008, Johnson was nominated for an ALMA Award for Outstanding Female Performance in a Comedy Television Series for her work on MADtv.

Johnson was given her own one-hour Comedy Central special, Anjelah Johnson: That's How We Do It, in 2009. She also appeared in the films Alvin and the Chipmunks: The Squeakquel and Our Family Wedding, and provided her voice for the live-action adaptation of Marmaduke. As well, she was featured on the late night talk show Lopez Tonight, hosted by George Lopez.

Johnson married Group 1 Crew member Manwell Reyes on June 11, 2011, in Half Moon Bay, California. She says the couple does not want children of their own, although they enjoy spending time with their nieces and nephews.

In 2013, Johnson reprised her role as her MADtv character Bon Qui Qui for a skit released by Alexander Wang. Johnson's second stand-up comedy special entitled Homecoming Show followed and premiered on Netflix. It was filmed in San Jose, California.

On October 1, 2015, Johnson's third stand-up special entitled Anjelah Johnson: Not Fancy was released on Netflix. It was filmed in Southern California.