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The series' protagonists are Lincoln Burrows (played by Dominic Purcell) and Michael Scofield (played by Wentworth Miller). Lincoln is a high school dropout and a convicted felon, who is wrongfully accused of and charged with the murder of Terence Steadman, the brother of the Vice President of the United States. Michael is Lincoln's brother and worked as a structural engineer before devoting full-time to his brother's case. In order to save his brother's life, Michael creates an elaborate plan to help his brother escape from prison. Over the course of the series, Michael and Lincoln are shown to be protective of each other. They are the only characters to have appeared in every episode to date.

Veronica Donovan (played by Robin Tunney) is Michael and Lincoln's childhood friend and decides to review Lincoln's case at Michael's insistence. She becomes Lincoln's lawyer and appears as a major character in the first season.

L. J. Burrows (played by Marshall Allman) is the teenage son of Lincoln Burrows and is greatly affected by his father's death sentence. He is forced into hiding after he becomes the target of the people who want Lincoln dead.

The two most prominently featured prisoners who Michael meets at Fox River State Penitentiary are Fernando Sucre (played by Amaury Nolasco) and Theodore "T-Bag" Bagwell (played by Robert Knepper). Sucre is Michael's cell-mate and provides comic relief to the show. His character's story focuses mainly on his wish to reunite with his girlfriend. Sucre develops a friendship with Michael and Lincoln, and becomes their ally. T-Bag is a cunning, violent and manipulative psychotic, consistently underestimated by those around him.

After he was incarcerated, John Abruzzi (played by Peter Stormare) became a prominent figure at Fox River State Penitentiary due to his role as the leader of a Chicago mafia. He agrees to provide an escape plane for Michael in exchange for the location of the eyewitness to his crimes, Otto Fibonacci. He appears regularly in the first half of the first season and makes selected appearances towards the end of the first season and the beginning of the second season. Benjamin Miles "C-Note" Franklin (played by Rockmond Dunbar) is introduced as a Fox River prisoner, who becomes suspicious of Michael. He later forces Michael to include him in the escape.

As well as other prisoners, Michael meets the captain of Fox River's correctional officers, Brad Bellick (played by Wade Williams) and the prison doctor, Sara Tancredi (played by Sarah Wayne Callies). Bellick dislikes Michael and causes trouble for him throughout the series. Sara is Michael's love interest. As their relationship develops over the course of the series, her role is also increased in the storyline.

Although Paul Kellerman (played by Paul Adelstein) was introduced as a Secret Service agent working for the Vice President to make sure that the execution of Lincoln Burrows goes smoothly, his character has alternated between a villain and an ally to Michael and Lincoln.

Season 2 introduces a new major character, FBI Special Agent Alexander Mahone (played by William Fichtner), who is assigned to locate the fugitives. Mahone is intellectually matched with Michael and his background unfolds as the series progresses. This led to the exit of Robin Tunney's character Veronica Donovan and Peter Stormare's character John Abruzzi at the beginning of the season. Sara Tancredi rises to become an extremely major character in Season 2. At the end of the season, Paul Adelstein's character exited, apparently assassinated.

After the exit of 3 main characters, Season 3 sees the addition of four major characters. Lechero (played by Robert Wisdom) is a prisoner at Sona and a Panamanian drug kingpin, James Whistler (played by Chris Vance) is incarcerated in Sona for the murder of the Mayor's son, Gretchen "Susan B. Anthony" Morgan (played by Jodi Lyn O'Keefe) is an operative for the company who is in charge of ensuring the escape of James Whistler and Sofia Lugo (played by Danay Garcia), who is Whistler's girlfriend. Sarah Wayne Callies' character Sara Tancredi was killed in the season's fourth episode. Wentworth Miller later commented on Callies' departure as a "loss" to the series.

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