This comes as rising stock markets swelled the ranks of billionaires, which included a record number of women.
With a net worth of $76 billion, the Microsoft Corp co-founder reclaimed the top spot after a four-year hiatus, toppling Mexico's telecommunications mogul Carlos Slim Helu, who placed second at $72 billion, Forbes said in announcing the list on Monday.
Amancio Ortega, the Spanish founder of clothing conglomerate Inditex SA, which includes the Zara fashion chain, ranked third at $64 billion.
Investing icon Warren Buffett, who runs Berkshire Hathaway Inc and is a frequent bridge partner for Gates, was fourth at $58.2 billion. Oracle Corp chief Larry Ellison came in fifth at $48 billion.
Gates has topped the list in 15 of the last 20 years.
A record 1,645 billionaires with a total net worth of $6.4 trillion made Forbes' list, up from 1,426 last year.
Just over 10 percent were female, with 172 women compared with 138 a year earlier.
Wal-Mart Stores Inc heiress Christy Walton was the highest-ranking woman, in ninth place, at $36.7 billion. France's Liliane Bettencourt, who got much of her wealth from cosmetics company L'Oreal SA, was next among women at $34.5 billion, and ranked 11th overall.
The Internet was well-represented. Google Inc founders Larry Page and Sergey Brin ranked 17th and 19th, worth a respective $32.3 billion and $31.8 billion, while Amazon.com Inc's Jeff Bezos was between them at $32 billion.
Facebook Inc founder Mark Zuckerberg, 29, more than doubled his net worth to $28.5 billion, and ranked 21st.
Former New York City Mayor Michael Bloomberg, who left office two months ago, was 16th at $33 billion, built mainly through his eponymous media company.
Forbes said the year's biggest loser was Brazilian tycoon Eike Batista, whose net worth fell below $300 million from $10.6 billion as his oil and natural resources empire collapsed amid too much debt and falling output.
Roughly two-thirds of the world's billionaires, or 1,080, were self-made. The United States had the most billionaires, with 492, followed by China at 152 and Russia at 111. Algeria, Lithuania, Tanzania and Uganda joined the list with one each.