Mathieu also ran track and field for St. Augustine's track team and was a member of the relay team. He was one of the state's top performers in the long jump. He had personal-best leaps of 1.93 meters in the high jump and 7.29 meters in the long jump.
Considered a four-star recruit by Rivals.com, Mathieu was listed as the No. 13 cornerback in the nation in 2010.
As a true freshman in 2010, Mathieu played in all 13 games with one start. He finished the season with 59 tackles, 3 sacks and 2 interceptions. He was the Most Outstanding Defensive Player of the 2011 Cotton Bowl Classic after he recorded seven tackles, one tackle for loss, two forced fumbles, one fumble recovery, one interception, one sack and one pass breakup.
At the end of the 2011 regular season, Mathieu had 77 total tackles (60 solo and 17 assists), 1.5 sacks, 5 forced fumbles, 4 fumble recoveries (2 returned for a touchdown), and 2 interceptions. On special teams, Mathieu had 26 punt returns for 420 yards, with 2 returned for a touchdown. On October 19, 2011, Mathieu was suspended for one game following a violation of LSU's drug policy. Mathieu was named the most valuable player of the 2011 SEC Championship Game. Mathieu earned the nickname "The Honey Badger" for his tenacious ability to play extremely tough football against much larger opponents, as well as his knack for making big plays. This led to him being a Heisman Trophy finalist. On December 8, 2011, Mathieu was awarded the Chuck Bednarik Award, given to the year's best defensive player in college football. This marked the second straight year a LSU defensive player won the nation's top defensive honor following Patrick Peterson, who also wore number 7, in 2010.
On August 10, 2012, LSU coach Les Miles announced that Mathieu would be dismissed from the football team due to a violation of team rules. News outlets reported that the dismissal was a result of repeated drug test failures, although Miles did not confirm those reports. Mathieu had formerly expressed an interest in returning to the program for the 2013 season. On August 17, 2012, Mathieu withdrew from LSU and entered a drug rehabilitation program in Houston, Texas. Mathieu's withdrawal was expected to last for the entire 2012-2013 academic year. However, on September 4, 2012, Mathieu ended his withdrawal and enrolled for fall semester classes. On October 25, 2012, Mathieu and three other LSU former players were arrested on possession of marijuana.
On November 29, 2012, it was announced that Mathieu would enter the NFL Draft. Mathieu participated at the 2013 NFL Scouting Combine on February 26, 2013. He ran a 4.50 40 Yard Dash, had 4 reps of 225 pounds, a 34-inch vertical, and a 117-inch broad jump. Tyrann would also later participate at his pro day on March 27, 2013.
In a Week 1 loss to the St. Louis Rams, Mathieu forced a fumble on tight end Jared Cook by knocking the ball out of his hand just before he entered the endzone allowing one of his teammates to recover the football. In a Week 2 win against the Lions, Mathieu made the game winning tackle when he stopped Nate Burleson short on 4th and 4. In the Week 3 loss against the New Orleans Saints, Mathieu intercepted a Drew Brees endzone pass for his first career interception. On December 8, Mathieu tore both his left ACL and LCL in a victory over the St. Louis Rams. On December 10, he was placed on the injured reserve, officially ending his rookie season.