Aylesbury MP Rob Butler's office said he would present the petition to Parliament at the earliest opportunity.
Mr Butler was presented with the petition, calling on the Department for Transport to give the line the green light, by Aylesbury councillor and chair of the Aylesbury Community Board, Mark Winn, who has been leading the local campaign for the railway since it emerged plans for the link were under threat.
The original plans for the East West Rail line included a spur running between Aylesbury and Milton Keynes, via the new Winslow Station.
But this is now under review.
Councillor Winn said: "This petition shows the strength of feeling amongst local people on the need for the Aylesbury link of East-West Rail.
"It would have a huge impact on the town, especially reducing the amount of traffic on the roads, which is the bane of residents' daily lives.
"More than that, the Aylesbury link would also benefit the entire county by providing a north-south line across Buckinghamshire.
"Rob has repeatedly raised the need for the Aylesbury rail link in the House of Commons and I'm delighted he'll now take this petition to Parliament to underline the importance of getting this train service in Buckinghamshire's county town.
"After all, this is the railway we do want locally - unlike HS2."
MP Rob Butler said: "The Aylesbury rail link is vital to the future prosperity of our town.
"As I've said throughout my time as an MP, development must benefit everyone and, as Aylesbury continues to grow, we need the right infrastructure to support it.
"The line would cut congestion on the roads, improve journey times on the rails, and reduce air pollution.
"I'd like to congratulate Mark for his work organising this petition, which I will be taking to Parliament.
"It will undoubtedly help me reinforce the arguments I have already made to the Department for Transport."
The Oxon and Bucks Rail Action Committee (OBRAC) encouraged its members to sign the petition.
Committee secretary Chris Wright said: "Some of the rail schemes being examined by the DfT cannot possibly match the benefits of East West Rail to Aylesbury, with all the connections on offer at Milton Keynes and journey time savings.
"It will be serving the growing Aylesbury area (90,000 people)and south Bucks and Milton Keynes (300,000 and plans to go to 500,000).
"Grant Shapps praised the £40 million rebuilding of the 10-mile route to Okehampton (population 7,500) which seems an odd priority in comparison."
Buckinghamshire Council's Transport, Environment and Climate Change Select Committee is due to receive an update on East West Rail on Thursday, January 20.
Buckinghamshire Council has committed £15.5 million into East West Rail, including some funding from Network Rail, on the assumption Aylesbury would be served.