The 27-year-old American was overtaken in the rankings two weeks ago by Dinara Safina, making the Russian and older sibling Marat Safin the only brother-sister duo to have been No. 1 in professional tennis.
"We all know who the real No. 1 is. Quite frankly, I'm the best in the world," Williams said on Monday, adding that she is her toughest opponent and "I always beat myself."
Safina has never won a Grand Slam title, losing last year's French Open final and being beaten in the Australian Open final this year by Williams.
"I'm excited for her," Williams said with a smile.
Williams was speaking at the Italian Open, which she won in 2002 and where she will play her first clay-court event of the season.
She opens against 2005 runner-up Patty Schnyder of Switzerland on Tuesday.
"I'm happy to be here," Williams said of the warmup for the French Open. "I think it will be a good event for me."
Her sister Venus Williams, seeded fourth and on the opposite side of the draw to Serena, is also in Rome and scheduled to play Monday against Lucie Safarova of the Czech Republic.
Safina, the 2006 runner-up, two-time defending champion Jelena Jankovic of Serbia and Ana Ivanovic of Serbia are among the top eight seeded players who have first-round byes.