The Pittsburgh Steelers selected Dupree in the first round (22nd overall) of the 2015 NFL Draft. He was the first University of Kentucky player taken in the first round since defensive tackle Dewayne Robertson went with the fourth overall pick to the New York Jets in the 2003 NFL Draft. He was the second linebacker selected in the draft, behind Vic Beasley who was selected with the eighth overall pick.

As a true freshman at University of Kentucky in 2011, Dupree played in 12 games with three starts at linebacker. During games against Georgia and Vanderbilt, he had a season high five tackles in each game. He finished the season with 21 tackles, 2.5 sacks, and one fumble recovery.

As a sophomore in 2012, Dupree started all 12 regular season games and recorded 91 tackles, 12.5 tackles-for-loss and 6.5 sacks. He recorded a season high 12 tackles against Western Kentucky. For his sophomore season he ranked 10th in the Southeastern Conference (SEC) in tackles and seventh in the FBS for sacks and tackles for loss. After this season he would switch from linebacker to defensive lineman for his last two years at Kentucky.

As a junior, in 2013, he played in 11 games, ranking second on his team with 61 tackles, first on the team with 9.5 tackles-for-loss at defensive end. He also had seven sacks and two forced fumbles. Against Mississippi State, Dupree recorded a career-high 13 tackles. In the SEC he finished sixth in sacks.

Dupree returned as a starter at defensive end his senior season in 2014 and was voted team captain for the Wildcats that year. For the season he had 74 tackles, 12.5 tackles-for-loss, and eight sacks. He also started all 12 games and recorded his first career interception which he returned for a game-winning touchdown in a 45-38 win against South Carolina. On Saturday, May 9, 2015, he graduated from the University of Kentucky with a degree in community and leadership development.

Coming out of college, Dupree was projected to be a first round draft pick by the majority of NFL analysts. He received an invitation to the NFL combine and completed the majority of combine drills. Dupree was unable to perform the bench due to a pectoral injury and opted to not run the short shuttle and three-cone drill after suffering a groin injury. On March 12, 2015, he participated at Kentucky’s pro day and performed very well in positional drills, the short shuttle, and three cone drills for scouts and teams representatives from 26 NFL teams. Minnesota Vikings’ head coach Mike Zimmer, Cincinnati Bengals’ head coach Marvin Lewis, and Pittsburgh Steelers’ head coach Mike Tomlin attended to scout Dupree, Za’Darius Smith, Braylon Heard, and 15 other prospects. Analysts and scouts gave him positive reviews for his freakish athleticism, explosive burst, large frame, outstanding power and strength, ability to play zone coverage, and character. The only criticisms he received were due to his below-average instincts and lack of consistency. He was also ranked the top defensive end out of the 148 available by NFLDraftScout.com, was ranked the fourth best outside linebacker by Sports Illustrated, the fifth best edge rusher by NFL analyst Charles Davis, and was ranked the second best edge rusher by NFL analyst Mike Mayock.

On May 15, 2015, the Steelers signed Dupree to a four-year contract worth $9.22 million with $8.63 million guaranteed and a signing bonus of $4.97 million.

He began his rookie season as the backup left outside linebacker behind veteran Arthur Moats. Dupree made his professional regular season debut in the Steelers’ regular-season opener against the New England Patriots and finished the 28–21 loss with two solo tackles and sacked Tom Brady. The next game, he made a season-high four combined tackles and a sack during the Steelers’ 43–18 victory over the San Francisco 49ers. On November 29, 2015, he made his first career start against the Seattle Seahawks and made one solo tackle. He finished his rookie season with 26 combined tackles, four sacks, and a pass deflection while playing in all 16 regular-season games and five starts.

Dupree was named the starting left outside linebacker ahead of Moats to begin the 2016 regular season. On September 4, 2016, Dupree was placed on injured reserve after suffering a groin injury. He was activated off injured reserve on November 19, 2016, prior to Week 11. He played in his first game of the season the following game. On November 24, 2016, he recorded two solo tackles in a 28–7 victory at the Indianapolis Colts. On December 11, 2016, he made his first start of the season and recorded a career-high five solo tackles, an assisted tackle, and sacked Bills’ quarterback Tyrod Taylor 2.5 times during a 27–20 victory over the Buffalo Bills.

Dupree was unable to play in the season-opener against the Cleveland Browns due to a shoulder injury. On September 17, 2017, he made his season-debut and collected two combined tackles, deflected a pass, and sacked Minnesota Vikings’ quarterback Case Keenum during a 26-9 victory. In Week 4, Dupree recorded four solo tackles and sacked Joe Flacco, as the Steelers defeated the Baltimore Ravens 26-9.

On April 23, 2018, the Steelers picked up the fifth-year option on Dupree’s contract worth $9.23 million On September 24, Dupree intercepted his first career pass off Ryan Fitzpatrick, and returned it for a touchdown. Dupree finished the 2018 season with 42 tackles, 5.5 sacks, 3 passes defended, and 1 interception returned for a touchdown. He received an overall grade of 60.5 from Pro Football Focus in 2018, which ranked as the 78th highest grade among all qualifying edge defenders.

In week 4 against the Cincinnati Bengals, Dupree forced a fumble off Andy Dalton in the 27–3 win. In week 9 against the Indianapolis Colts, Dupree recorded two sacks on Brian Hoyer, one of which was a strip sacked that he forced and recovered, in the 26–24 win, earning AFC Defensive Player of the Week honors. In week 12 against the Cincinnati Bengals, Dupree recorded a strip sack on Ryan Finley and recovered the football in the 16–10 win. In the following week’s game against the Cleveland Browns, Dupree sacked Baker Mayfield 1.5 times, one of which included another strip sack that was recovered by Cam Heyward, in the 20–13 win. Dupree had a career year in 2019 recording 11.5 sacks, 4 forced fumbles, and 68 combined tackles.

The Steelers placed the franchise tag on Dupree on March 16, 2020. He signed the one-year tender on April 23, 2020.