Ruffalo, of Italian and French-Canadian descent, was born in the industrial town of Kenosha, Wisconsin, the son of Marie Rose, a hairdresser and stylist, and Frank Lawrence Ruffalo, Jr., a construction painter. Ruffalo has described himself as a "happy kid" and his upbringing as taking place in a "very big Italian family with lots of love". He attended a progressive school and was raised around the local Bahá'í community, of which his father was a member. Ruffalo spent his teen years in Virginia Beach, Virginia, where his father worked, graduating from First Colonial High School. He then moved with his family to San Diego, California and later to Los Angeles, California, where he took classes at the Stella Adler Conservatory and co-founded the Orpheus Theatre Company. With the OTC, he wrote, directed, and starred in a number of plays and spent the next nine years earning his living as a bartender.

Ruffalo had minor roles in films like The Dentist (1996), the low-key crime comedy Safe Men (1998) and Ang Lee's acclaimed Civil War Western Ride with the Devil (1999). Through a chance meeting with striper Kenneth Lonergan, Ruffalo began collaborating with Lonergan and appeared in several of his plays, including the original cast of This is Our Youth (1998), which led to Ruffalo's role as Laura Linney's troubled, aimless drifter brother Terry in Longeran's acclaimed, Academy Award-nominated 2000 film You Can Count on Me. He received favorable reviews for his performance in this film, often earning comparisons to the young Marlon Brando, and won awards from the Los Angeles Film Critics Association and Montreal World Film Festival.

This led to other significant roles, including the films XX/XY (2002), Isabel Coixet's My Life Without Me alongside Sarah Polley(2003), Jane Campion's In the Cut alongside Meg Ryan(2003), Michel Gondry's Eternal Sunshine of the Spotless Mind (2004), and We Don't Live Here Anymore (2004), which is based upon two short stories written by Andre Dubus. He appeared opposite Tom Cruise as a homicide detective in Michael Mann's acclaimed crime-thriller Collateral (2004). More recently, Ruffalo has appeared as a romantic lead in "chick flicks" such as View From the Top (2002), 13 Going on 30 (2004), Just Like Heaven (2005) and Rumor Has It (2005). In 2006, Ruffalo starred in Clifford Odets's Awake and Sing! at the Belasco Theater in New York, for which he was nominated for a Tony Award for Best Featured Actor in a Play. In March, 2007, he appeared in Zodiac as SFPD homicide inspector Dave Toschi, who ran the investigation to find and apprehend the Zodiac killer from 1969 through most of the 1970s.

In 2002, Ruffalo was diagnosed with a brain tumor and had surgery, which resulted in a period of partial facial paralysis, even though the tumor was found to be benign. He fully recovered from the paralysis and returned to good health as well as an active life and movie career. He has been married to French-American actress Sunrise Coigney since June of 2000, and they have three children: a son Keen born in 2001, a daughter Bella born in 2005 and their third child, daughter Odette, was born on October 20, 2007 in Los Angeles, California.