The 'Born This Way' hit maker - who joined the social networking site in 2008 - has beaten Justin Bieber, who is languishing behind with 18.1 million followers, to the milestone with her @ladygaga account.
Gaga - whose real name is Stephanie Germanotta - uses the account, which she started on March 26, 2008, to interact with her supporters, who she refers to as Little Monsters.
Since overtaking Britney Spears as the most-followed person on the website in August 2010, the singer has held onto the top spot.
Gaga is well ahead of her fellow singers Katy Perry, who has 15.5 million followers, as well as Shakira and Rihanna, who both have more than 14.5 million.
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Despite reaching the milestone, the 'Paparazzi' singer hasn't tweeted since March 3 - but she has previously used the account to announce benefits for her fans.
In December 2011, Gaga revealed she was going to make a free download of an unreleased track available as a Christmas present to her supporters.
She tweeted: "Been racking my brain on what to get Little Monsters for Christmas!! I finally figured it out!! (sic)
"On Christmas Day I will leak to you an unreleased Song off Born This Way! It was recorded live, in one take, on the tour bus. Uncensored (sic)."
As well as her large following on twitter, Gaga has eight million fans on Facebook and more than 800,000 'circles' on Google+.