The 29-year-old actor has been suggested as a possible replacement for Daniel Craig as 007 but his Marvel contract is said to forbid him from taking any parts that could overshadow his lead role in the Spider-Man movies.
An insider told The Sun on Sunday newspaper's Bizarre column: "Tom can't play two superheroes, it just won't happen."
Holland previously suggested that he was open to playing Bond and declared that landing the role would be the "pinnacle" of his career.
Speaking to Gordon Ramsay on YouTube, he said of the rumours: "Listen, there's speculation at the moment. We'll keep it to a minimum for now. We'll get there one day."
The Hell's Kitchen star then further pressed Tom on the issue.
He said: "Can you imagine what's going to happen to your life if you do? First of all, you'd love that opportunity, right?"
Tom replied: "I mean every young British actor, it's the pinnacle of working in our industry. I already consider myself to be the luckiest kid alive. I could not have dreamed to have the career that I have."
The 26th Bond film, directed by Denis Villeneuve, is set to head into production at Pinewood Studios in the UK in April next year and the story - which is being written by Peaky Blinders creator Steven Knight - will centre on the threat posed by artificial intelligence (AI).
The source said: "This is the first Bond of the AI era and the plot is going to reflect that. Writers are going to have some fun.
"This next Bond movie has to feel bold, fresh and relevant - especially after such a long break.
"There's a sense of cautious excitement behind the scenes. They want to modernise Bond without losing that classic swagger - and that's a tricky balance.
"Denis isn't coming in to make a safe Bond film - he's coming into redefine what Bond can be. Think scale, style and substance."
The new movie will mark a new era for the franchise after Amazon MGM took creative control from long-time producers Barbara Broccoli and Michael G Wilson earlier this year but Villeneuve vowed to "honour the tradition" of the iconic spy when he was announced as the director of the latest movie.
The 58-year-old filmmaker said: "Some of my earliest movie-going memories are connected to 007. I grew up watching James Bond films with my father, ever since Dr. No with Sean Connery.
"To me, he's sacred territory. I intend to honour the tradition and open the path for many new missions to come."
Amazon MGM Studios chief Mike Hopkins added: "Denis has delivered compelling worlds, dynamic visuals, complex characters, and - most importantly - the immersive storytelling that global audiences yearn to experience in theatres.
"James Bond is in the hands of one of today's greatest filmmakers and we cannot wait to get started on 007's next adventure."
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