He made his debut in 2001 with the blockbuster Mujhe Kucch Kehna Hai, a remake of the Telugu super hit Tholi Prema, along with Kareena Kapoor. He received the Filmfare Award for Best Male Debut for his work in the film. He then starred as Rahul in coming of age film Kyaa Dil Ne Kahaa alongside Esha Deol. Kapoor then appeared in two more Telugu remakes Jeena Sirf Merre Liye (2002), and Yeh Dil (2003), which fared moderately well at the box office.

He then appeared in Ram Gopal Varma's production Gayab (2004) which had kapoor receiving appreciation for his work. Starting 2004, Kapoor was starred in commercial hits such as Khakee (2004), Kyaa Kool Hai Hum (2005), Golmaal (2006), Shootout at Lokhandwala (2007) in which he has essayed the role of gangster Dilip Buwa, and received good reviews for his work, Golmaal Returns (2008), Golmaal 3 (2010), The Dirty Picture (2011), Kyaa Super Kool Hain Hum (2012), the critically acclaimed Shor in the City (2012), and Shootout at Wadala (2013) .

In 2012, Kapoor took part in the "Fashion for a Cause" event which raised money for homeless children. He has co-produced the film Chaar Din Ki Chandni (2012), in which he has also starred. He worked as a lead actor in Bollywood adult comedies Kyaa Kool Hain Hum 3 (2016) and Mastizaade (2016) both did average Business.

Tusshar Kapoor is the son of celebrated Bollywood actor Jeetendra and of Shobha Kapoor. His sister Ekta Kapoor is a prominent television and film Producer. He attended the Bombay Scottish School where he was in the same class as Abhishek Bachchan. He then studied at the University of Michigan in Ann Arbor for his BBA degree at the Stephen M. Ross School of Business. Like his father, Kapoor is a religious Hindu, and he practices Khatri Hindu Khatri Kapoor opted for IVF and became single parent to Laksshya Kapoor through surrogacy in June 2016.

In 2017, he starred as Lucky in the comedy film Golmaal Again, which ended up being a blockbuster.