Madhoo was born as Madhoobala Raghunath Malini on 26 March 1969 in Madras (now Chennai), Tamil Nadu, India. She comes from a Tamil family and is related to veteran actress Hema Malini. She studied in Mumbai and was trained in Bharatanatyam by her mother.
Madhoo began her film career in the early 1990s. Her first released film was the Malayalam movie Ottayal Pattalam (1991), while she made her Hindi-film debut with Phool Aur Kaante (1991) opposite Ajay Devgn.
Her major breakthrough came with Mani Ratnam's Tamil film Roja (1992), in which she played Roja, opposite Arvind Swamy. The film became a major success and Madhoo's performance brought her nationwide recognition.
She subsequently appeared in successful films across several Indian languages, including Allari Priyudu (1992), Yoddha (1992), Gentleman (1993), Pehchaan (1993), Diljale (1996), Yeshwant (1997), and Thalaivii (2021). Her multilingual career made her a recognised face in both South Indian and Hindi cinema.
Madhoo later returned to prominent screen work after a period away from mainstream films. She has continued appearing in films and television/streaming projects, including Shaakuntalam (2023), Sweet Kaaram Coffee (2023), Prem Kumar (2023), and Game On (2024).
She married Anand Shah in 1999, and they have two daughters. Madhoo continues to remain active in the Indian entertainment industry.