In 1961, Lee created The Fantastic Four for Marvel Comics, and went on to create titles including Spider-Man and The Incredible Hulk.
The legendary comic book author died at Cedars Sinai Medical Center in Los Angeles, according to a family lawyer.
The CEO of Lee's Pow! Entertainment praised the "father of pop culture" for inventing "universes of characters".
CEO Shane Duffy called him "a true iconic pioneer with no comparable second".
Lee's wife, Joan, died in 2017 - also aged 95 - but he is survived by his daughter, JC Lee.
Speaking to celebrity news website TMZ, JC Lee said her father was "the greatest, most decent man".
She told Reuters: "He felt an obligation to his fans to keep creating."
In recent years, Lee had periodically suffered from illnesses, including a bout of pneumonia, US media reported.