Ambani, whose fortune has fallen for the third time in a row, beat London-based steel baron Lakshmi Mittal and Wipro Chairman Azim Premji to top the list.
Despite a slowing economy, the collective wealth of the richest 100 Indians grew at 3.7 percent to a total of $250 billion.
The top 10 has familiar faces such as Pallonji Mistry, Adi Godrej, Savitri Jindal, Shashi and Ravi Ruia, Hinduja Brothers and Kumar Mangalam Birla.
The only exception is Dilip Sanghvi, who entered the top five for the first time with a net worth of $9.2 billion, boosted by the shares of his Sun Pharmaceuticals Industries.
While Sanghvi was the biggest dollar gainer, his fellow entrepreneur, Wockhardt's Habil Khorakiwala, was the biggest percentage gainer with a worth of $1.8 billion.
A McKinsey report earlier this year projected India's pharmaceutical market would triple to $20 billion by 2015 and move into the world's top-10 pharmaceutical markets.
Forbes said there were 61 dollar billionaires in the country in 2012, up by four from the previous year.
Copyright Thomson Reuters 2012