John Popper was born in Chardon, Ohio. His father was a Hungarian immigrant who left Budapest in 1948. Through him, Popper is related to David Popper, a 19th-century European Cellist whose many solo works for the cello are staples of the instrument's repertoire. John's mother and brother are lawyers.

Popper was raised in Stamford, Connecticut, New York, and New Jersey. He attended Davenport Ridge School, Stamford Catholic High School (now Trinity Catholic High School) and Princeton High School from which he graduated in 1986. He took lessons on the piano, the cello, and the guitar, but none of those instruments appealed to him and he hated being forced to practice.

He formed several garage bands with friends in Princeton, New Jersey, one of which evolved into Blues Traveler in 1987. After graduating from high school, the group's members all moved to New York City, where Popper enrolled in The New School for Jazz and Contemporary Music along with two of his bandmates and high school friend Chris Barron. Popper attended for three years but devoted himself to the band full-time once they signed a record contract in 1990.

In 1990, Popper began to perform occasional solo concerts in addition to touring with Blues Traveler. Several songs that originated as Popper's solo pieces have become part of Blues Traveler's repertoire, and vice versa.

In October 1992 Popper was involved in a traffic accident on a motorcycle while traveling to a studio to record for Blues Traveler's third album. The accident put him in a wheelchair for several months, but Popper continued touring with the band despite the difficulties it created.

Popper had a speaking guest role in an episode of the sitcom Roseanne as a musician similar to himself. Popper appeared on Episode 30 of Space Ghost Coast to Coast as a musical guest. Popper appeared as a guest on the IFC television comedy series Z Rock. He plays a role as himself, and befriends the band ZO2, helping them get a record deal. He also played the Star-Spangled Banner prior to Game 4 of the 1996 World Series.

Popper was a part of a one-time studio band brought together in 1997 by New York drummer/songwriter Solomon Deniro. Other players included Trey Gunn, Bernie Worrell, Marc Ribot, and Vernon Reid. The group's only recordings were released as the album Gimme Gimme under the name the Devotees. The same recording was re-released by Deniro in 2001 with the title Solomon.

Popper took over in 1998 as front man of jam-band supergroup Frogwings, which released the live album Croakin' at Toad's. Frogwings was mainly active until 2000.

In 1999, he suffered a near-fatal heart attack brought on by years of compulsive overeating. (He had been diagnosed with diabetes a few years earlier.) Doctors at Cedars-Sinai Medical Center performed an emergency angioplasty which saved Popper's life; he had 95% arterial blockage. Popper later underwent gastric bypass surgery and lost a significant amount of weight.

Popper has toured with the USO, both with Blues Traveler and solo. In the mid-2000s, he toured the Middle East, performing with the Band of the Air Force Reserve and Jamie O'Neal at various military camps.

Popper has developed some equipment innovations to accommodate his use of harmonicas during onstage performances. Because each individual diatonic harmonica is tuned to one particular key, he fashioned belts with enough pockets to hold harmonicas in all 12 keys (plus extras) and wore them as a bandolier, or slung over his neck. He frequently has to switch keys multiple times within one song, and this arrangement allowed him to quickly trade one harmonica for another without looking. In 2002, he stopped using the belts because they no longer fit him properly due to his weight loss it sold on pawnstars for $2700; now he carries his harmonicas in a small black attaché case. He uses a special microphone with switches that change the audio effect of the harmonica as it is played through an amplifier, similar to a guitar effects pedal. Popper was inspired by Jimi Hendrix's guitar playing to make his instrument sound however he wanted. He has fashioned a number of floppy-brimmed hats with flattened harmonica cover plates on the band, which he almost always wears during appearances with Blues Traveler.

Popper is a member of the Libertarian Party, and has previously expressed support for the Republican Party. He endorsed George W. Bush in the 2004 U.S. presidential election. In November 2008, Popper said, regarding Barack Obama, "this is the first time I've voted for a Democrat, ever." John Popper was a supporter of Ron Paul during the 2012 U.S. Presidential election, even participating in phone-banking at Ron Paul's New Hampshire campaign headquarters. He also played a short set during Ron Paul's "We are the Future Rally," an alternative convention for Paul supporters which was held in Tampa the day before the 2012 Republican National Convention.

Recently, Popper formed a rock/jazz/hip-hop fusion group the John Popper Project with DJ Logic, which released an album in 2006 and performs occasionally. He also performs on the album Global Noize by Jason Miles and DJ Logic (2008).

In 2003, Popper was arrested for possession of marijuana. Popper was arrested again on March 6, 2007 near Ritzville, Washington by the Washington State Patrol. He was the Passenger in his own vehicle, which was stopped for speeding, and was found to be in the possession of a small amount of marijuana and weapons. Popper was released the same night. The vehicle had a stash of hidden compartments which contained four rifles, nine handguns, a switchblade knife, a Taser, a set of brass knuckles, and night vision goggles. The vehicle was temporarily seized.

Popper has co-written songs with Trey Anastasio, Warren Haynes, Chris Barron, and Jonny Lang. He also frequently appears as a guest performer with Musicians both famous and obscure, from a diverse variety of genres. He has performed with, among others, jam bands Spin Doctors, Dave Matthews Band, Phish and most recently, the Allman Brothers Band in 2009; bluesmen Eric Clapton and B.B. King; singer-songwriters Jason Mraz and John Mayer; Saxophonist Karl Denson; San Francisco's Culann's Hounds; heavy metal band Metallica; rock trio ZO2 and even with the Hungarian Ambassador to the United States, András Simonyi. He sat in with the Smashing Pumpkins on the second day of their acoustic 1997 Bridge School Benefit appearance, contributing harmonica for their song "Porcelina of the Vast Oceans"; Popper's solo garnered major applause from the audience. He also performed with the Grateful Dead at a tribute to Bill Graham in 1991. He was the harmonica player on Hanson's album This Time Around in 2000, for which he performed on "If Only" and "In The City."

He has been a recurring guest on Howard Stern's and Bill Maher's shows and sits in with the CBS Orchestra on the Late Show with David Letterman on occasion. In 2009, he sat in with the Roots on an episode of Late Night with Jimmy Fallon. Popper performed "Something Sweet" with the Duskray Troubadours on the TBS show "Lopez Tonight" on March 1, 2011. He also sat in with the house band for the closing number of the show.

John Popper has expressed a preference for the Hohner Special 20 brand blues harp, calling them "the Porsche of harmonicas." Since 2015 he uses mainly his own signature harmonicas manufactured by Fender. Popper uses Shure microphones and Mesa Boogie amplifiers, similar to bandmate Chan Kinchla. He also uses D'Addario strings.

Most recently, Popper performed his hits "Run-Around" and "Hook", as well as a rendition of "The Star Spangled Banner", alongside Greensky Bluegrass for the Bernie Sanders: A Future to Believe In rally at Safeco Field in Seattle on March 25, 2016.