CSI: NY follows a group of crime scene investigators (CSI's for short) who work for the New York Police Department's (NYPD for short) crime lab. The series mixes gritty subject matter and deduction in the same manner as its predecessors, yet also places a great deal of emphasis on criminal profiling. The team is led by Detective Mac Taylor, a former Marine from Chicago. Mac, a veteran of the NYPD, lost his wife on 9/11, and as such must work to rebuild his personal life while supervising his team. He is organized, efficient, dedicated, and very proper in his management style. Mac's partner is originally Detective Stella Bonasera. Stella is half-Greek, half-Italian. She helped Mac through the impact of his wife's death and has been by his side ever since. She is a savvy investigator, yet she often speaks before she thinks. Stella leaves New York to head a crime lab in New Orleans and is replaced by Detective Jo Danville. Jo is a former FBI criminalist and an experienced psychological profiler. She and Mac quickly form a strong friendship and an even stronger working rapport. Jo is still haunted by her ousting from the FBI after blowing the whistle on improper lab procedure, so she works to regain her professional reputation. Together, Mac, Stella, and Jo head an elite team of detectives including Detectives Danny Messer, Aiden Burn, and Lindsay Monroe. The team also works alongside former medical examiner-turned-CSI Sheldon Hawkes, Detective Don Flack, Medical Examiner Sid Hammerback, and CSI trainee Adam Ross.

• Mac Taylor (Gary Sinise) is a Detective First Grade and the director of the NYPD crime lab. Born in Chicago, Mac served as a major in the USMC, serving in both the 1983 Beirut barracks bombings and Operation Desert Storm, once saying that he had wanted to serve his country more than anything else in the world. Mac lost his wife, Claire, in the World Trade Center attacks, dates occasionally, and eventually proposes to an old friend, Christine Whitney, in the series finale.
• Stella Bonasera (Melina Kanakaredes) is a half-Greek, half-Italian orphan. She is a Detective First Grade with the NYPD and the Assistant CSI Supervisor of Mac's team. She often speaks before she thinks, leading to numerous complaints being made against her by those suspected of crimes. Never married and with no children, various episodes suggest she was subtly interested in Detective Taylor, but the latter was always shown to be grieving the death of his wife during the 9-11 terrorist attacks. Always going where she can do the most good, Stella resigns from the NYPD crime lab following the season-six finale to run the NOPD crime lab. (seasons 1–6)
• Danny Messer (Carmine Giovinazzo) is a Detective Third Grade and a Crime Scene Investigator working on Mac's team. Growing up in a family under surveillance Danny formulated his own set of hybrid ethics, blending the world of lawbreakers with the world of lawmakers. He is driven by ethics and one day hopes to run the New York crime lab, at one point taking a promotion to sergeant to put him on track to do so. He develops a romantic relationship with Lindsay Monroe.
• Aiden Burn (Vanessa Ferlito) is a Brooklyn native and a Detective Third Grade assigned to Taylor's team. In the first season she is the newest member of the CSI team, but is just as capable as any of them. She is fired from the crime lab after considering planting evidence on a rape suspect. Aiden goes on to apply for a private investigator's license and continues to investigate this suspect. She is fatally beaten and set on fire as a result, leaving the entire team devastated. During her murder, she was able to leave behind enough evidence to convict her killer. (seasons 1–2; guest star: season 2)
• Sheldon Hawkes, M.D. (Hill Harper) is a medical examiner with the NYC Office of the Chief Medical Examiner (OCME) when the series starts. He was a child prodigy who graduated from college at age 18 and was a board-licensed surgeon by the age of 24 and had several years experience in the emergency room. He left surgery because the pain of losing two patients was too intense. In the second season he becomes a CSI on Mac's team, although he is not a sworn NYPD detective like his colleagues.
• Don Flack (Eddie Cahill) is an NYPD Homicide Detective First Grade who comes from a long line of law enforcement officials. He bridges the gap between old-school NYPD and the new generation of CSI. His techniques are borderline yet effective. Despite being an NYPD strongman Flack is emotionally mature. He is left devastated when girlfriend Detective Jessica Angell is shot and killed on the job. Years later, he begins a relationship with Detective Jamie Lovato.
• Lindsay Monroe Messer (Anna Belknap) is a Detective Third Grade. She worked as a CSI in Montana and finally realized her dream of moving to a big city like New York. Her northwestern work ethic and willingness to tackle any job is a welcome addition to the team. She develops a romantic relationship with Danny Messer and the two marry in season five. Lindsay is awarded the Police Combat Cross by the NYPD for killing a psychopathic fugitive who had intruded her home. (seasons 2–9)

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The theme song for CSI: NY was taken from the first verse of The Who's "Baba O'Riley." For season four (and onward), the theme was revamped to be more instrumental, removing the lyrics: "I don't need to fight to prove I'm right. I don't need to be forgiven." The score composer for the series was Bill Brown, who won a BMI Award for season one.