The 21-year-old Portugal international midfielder joins from West Ham United.
Fernandes was eager to get started at his new club.
"I'm very excited for this next step. Spurs is a massive club and the Head Coach was a key part of why I have decided to join.
"When we spoke, it was very special. We look at football in the same way — going onto the pitch as a strong team, with fight and energy, to try and win every game. I can't wait to get started, to meet the fans, to meet everyone, and give everything for the Club."
Manager Roberto De Zerbi has long admired the midfielder's game.
"I've admired Mateus for a long time because he combines quality on the ball with the intensity and intelligence that are so important in the way we want to play.
"Despite his age, he already has good experience in the Premier League and has shown quality and consistency at this level. Mateus is comfortable under pressure, can progress the ball, works hard for the team and has the courage to make things happen in difficult moments."
Fernandes has gained valuable Premier League experience over the past two seasons, first at Southampton and then with West Ham United last term.
Fernandes ranked in the top ten for duels won, with 208, across 36 Premier League appearances for Saints.
He produced six goal contributions, two goals and four assists, including a stoppage-time strike in a 3-1 defeat at Tottenham Hotspur Stadium in 2024/25.
A constant presence in midfield throughout his debut season in England, Fernandes was named Southampton's Player of the Season before joining West Ham at the end of August 2025.