The 59-year-old actor and presenter has launched a petition to prevent buildings being demolished to make way for a new station.
A planned Crossrail 2 connection at Tottenham Court Road will see two new access points built in Soho.
Fry, who is chairman of Save Soho, warned the area "could change dramatically".
He said the group did not oppose Crossrail 2.
But he said: "Soho is not just a metropolitan enclave... it is a focus, a magnet for the young, creative and open-minded around the country.
"The new threat to Soho's authentic soul coming from Crossrail 2 is a real and urgent issue."
London Mayor Sadiq Khan has warned that London's transport network will grind to a halt due to overcrowding unless the government backs Crossrail 2.
Construction could start in the early 2020s and the railway could be open by 2033.
A preferred route for a proposed Â£20bn Crossrail 2 scheme, running north-south through London was approved in 2014.
Crossrail 2 bosses say improvements will reduce crowding on the busiest part of the Northern line during peak periods by approximately 30%.

The radio and radar station set up at Aird Uig, Gallan Head, 60 years ago was part of Nato's early warning system against Soviet submarines and aircraft.
Gallan Head Community Trust bought the site from the Ministry of Defence with help of grant funding.
It is working on turning parts of the site into a space observatory.
A marine research centre is also planned at what is to be called The Cetus Observatory.
The trust said: "Cetus will be a ground-breaking, multi-purpose space for observing the dark skies, changeable weather, listening to and spotting whales and getting absorbed in every aspect of nature and science.
"The observatory will hold an internet operated robotic telescope, a small radio telescope, a small radar, a solar telescope, a planetarium, a space exhibition, giant wide field binoculars for viewing cetaceans and birdlife, nature webcams, a cafÃ©, educational facilities, and a small shop.
It added: "Outside the centre there will be a circular path around the headland, in some places leading to observation shelters, excellent for daytime marine life spotting walks or night-time star gazing."
Stephen Fry has said Crossrail 2 poses a threat to Soho's "authentic soul".
[NEXT_CONCEPT]
A ceilidh and a torchlight procession are to be held on Saturday to mark a year since a community buyout of a Cold War surveillance station on Lewis.