Mr Gates' net worth rose by just over $3bn (£1.94bn) in the year to 13 February, to $79bn.
There are a record 1,826 billionaires in the world, Forbes said, an increase of 181 in the past 12 months.
Mr Gates has now been top of the list for 16 of the last 21 years, Forbes added.
Legendary US investor Warren Buffett regained third place in the list with a net worth of $72.7bn, from Amancio Ortega, the founder of Spanish fashion chain Zara.
1. Bill Gates $79.2bn (Microsoft)
2. Carlos Slim Helu $77.1bn (Phones and construction in Mexico)
3. Warren Buffett $72.7bn (Global investor)
4. Amancio Ortega $64.5bn (Zara and other fashion chains)
5. Larry Ellison $54.3bn (Oracle data storage technology)
6. Charles Koch $42.9bn (Industrialist)
7. David Koch $42.9bn (Industrialist)
8. Christy Walton $41.7bn (Walmart retail giant)
9. Jim Walton $40.6bn (Walmart retail giant)
10. Liliane Bettencourt $40.1bn (L'Oreal cosmetics firm)
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