He played college football for the Miami Hurricanes, and was selected by the Colts in the first round of the 2001 NFL draft with the 30th overall pick.

On December 14, 2014, Wayne played in both his 209th game and his 142nd win as a member of the Colts, breaking the franchise records set by Peyton Manning.

[3][4] A New Orleans Saints fan in his youth, he was more interested in baseball growing up but chose football by the time he attended John Ehret High School.

[9] He is one of only five wide receivers in school history to post 20 or more touchdowns in his career, along with Michael Irvin, Lamar Thomas, Leonard Hankerson, and Andre Johnson.

Wayne also ran track and field at the University of Miami, where he recorded a personal best of 21.87 seconds in the 200 meters.

Wayne graduated with a degree in liberal arts; his roommate was future Baltimore Ravens safety Ed Reed.

[41] In Week 17, against the Arizona Cardinals, the 13–2 Colts rested most of their top players in preparation for the playoffs, and Wayne had zero receptions for the last time in his remaining 134 regular season games.

[44] In the playoffs, Wayne had 97 yards, a two-point conversion, and a critical 22-yard reception to set up a potential game-tying field goal in a 21–18 loss to the eventual Super Bowl champion Pittsburgh Steelers in the Divisional Round.

[54] In the Divisional Round against the Baltimore Ravens, he had five receptions for 51 yards in the 15–6 victory where all of the Colts' points came from field goals.

[56][57] He helped the Colts defeat the Chicago Bears in Super Bowl XLI with a 53-yard touchdown reception in the first quarter.

[64] In 2007, Wayne responded to a rash of injuries on the team, including Harrison and tight end Dallas Clark, by setting a then-career-high in receptions (104), and a career-high in yards (1,510).

[65] He led the league in receiving yards and was selected to go to the Pro Bowl for the second consecutive year.

[73] In the Colts' 2009 season opener against the Jacksonville Jaguars, Wayne had ten receptions for 162 yards and a touchdown in the 14–12 victory.

In Super Bowl XLIV, Wayne had five catches for 46 yards, but the Colts lost 31-17 to the New Orleans Saints.

[83][84] During a season where the Colts heavily relied on Peyton Manning to win games, Wayne again cemented himself as one of the top receivers in the NFL.

[89] Wayne's numbers were lower than usual in 2011 without Peyton Manning starting at quarterback, though he still led team in receptions (75) and receiving yards (960).

[93] Before the Colts' Week 5 matchup with the Green Bay Packers, the news of head coach Chuck Pagano's leukemia surfaced.

He also passed former Washington Redskins receiver Art Monk for 12th on the NFL's all-time receptions list during the first half.

[99] During the Wild Card Round against the Baltimore Ravens, Wayne had 114 yards on nine receptions and moved into second in career playoff catches with 92–59 behind leader Jerry Rice.

[101] Wayne was selected as the wide receiver for USA Football's 2012 All-Fundamentals Team, which honors 26 NFL players each year for executing the fundamentals of their position.

[104] However, a week later, Wayne tore his ACL during a 39–33 victory over Denver, in Peyton Manning's first bout against the Colts since leaving Indianapolis in 2012; it was announced the next day that he would miss the remainder of the 2013 season.

The injury also ended his consecutive games played streak at 189, the third-longest for a wide receiver in NFL history.

[109] Near the conclusion of the Colts' 2014 season, it was announced that Wayne had played with a torn triceps since Week 6, which would require an offseason surgery to repair.

[119] On January 15, 2016, after not playing the entire 2015 season, Wayne announced his retirement from professional football, saying, "It was fun, but it's time.

[124] On November 18, 2018, Wayne became the 15th player to be inducted into the team's Ring of Honor, in a ceremony attended by former teammates including Marvin Harrison, Manning and Saturday.

[125] On January 2, 2020, Wayne was named one of 15 modern-era finalists for election into the Pro Football Hall of Fame in Canton, Ohio.