CSI: Miami follows a group of detectives assigned to the Miami-Dade Police Department's Crime Scene Investigations, an elite unit operating out of the (fictional) "Miami Dade police headquarters, with its eerie blue light and flickering screens".

The team is led by Lieutenant Horatio Caine (David Caruso), who, through his history as a bomb-disposal expert, has gained specialized knowledge in explosive forensics. Horatio believes that "evil is" and lives "between the perpetrators of this evil and the people who try and come between that evil and the citizen". In his pursuit of justice, he has proven that "he can handle himself on the street and he's not a person to be messed with".

The New York Sun has described Caine as an amalgam of "the spirits of all the laconic American law men who preceded him", while the New York Post describes Caine's partner Detective Calleigh Duquesne (Emily Procter) as "a bilingual Southern beauty with a specialty in ballistics".

Together, Caine and Duquesne head a team of forensic investigators that includes Lieutenant Megan Donner (Kim Delaney), conceived as "a strong woman [who could] duplicate the chemistry that Caruso displayed with Marg Helgenberger" during "Cross Jurisdictions", Detective Eric Delko (Adam Rodriguez), an underwater recovery expert, Walter Simmons (Omar Miller), a detective who forces the "CSIs to do more science and research instead of relying on databases", Los Angeles Police transfer Jesse Cardoza (Eddie Cibrian), former FBI agent Natalia Boa Vista (Eva LaRue), and Ryan Wolfe (Jonathan Togo), a master of genetics recruited following the death of Detective Timothy Speedle (Rory Cochrane). The team are assisted by Medical Examiner Alexx Woods (Khandi Alexander), who began her career as a medical Examiner in New York, and her replacement Tara Price (Megalyn Echikunwoke), Miami Dade Police Sergeant Frank Tripp (Rex Linn), and Horatio's sister-in-law, Detective Yelina Salas (Sofia Milos).

During their investigations, the team cooperate with both allies and nemeses, including Internal Affairs Lieutenant Rick Stetler (David Lee Smith), States Attorney Rebecca Nevins (Christina Chang), Medical Examiner Tom Loman (Christian Clemenson), and newly minted detective Sam Owens (Taylor Cole).