A group of more than 70 leaders from across the South East have written to chancellor Philip Hammond and transport secretary Chris Grayling, saying the infrastructure project is of "national importance".
"We need to get people to work," said the letter.
"Now is the moment for government to find the parliamentary time and show the world UK is open for business post Brexit."
The mayor of London, Sadiq Khan, who expressed disappointment at the lack of mention for Crossrail 2 in Hammond's Budget, said:
This is yet more evidence of just how important Crossrail 2 is to the UK.
Despite the project benefiting the whole of the UK, London has actually met half the funding cost. What we now need is for the government to take note of these benefits and give us the green light to progress, for the good of the entire country.
Crossrail 2 will connect National Rail networks in Surrey and Hertfordshire with an underground tunnel beneath central London between Wimbledon and Tottenham Hale and New Southgate.
Transport for London (TfL) has submitted an updated business case and funding plan to Chris Grayling and said the project will enable 270,000 more people to enter central London every morning at peak time.
A government spokesperson said: “We have now received the Strategic Outline Business Case from Transport for London and will carefully consider it to ensure it is robust and includes a fair, sustainable and deliverable funding plan.”
Proponents say its delivery will support the UK's engineering, construction and manufacturing sectors, including £1bn to the West Midlands economy, more than £900m to Yorkshire and Humber and up to £170m to the Scottish economy.
Grayling is due to make a decision in the spring on further government support. If it is given backing, construction could start in the early 2020s and the railway could be operational by 2033.
Business leaders have warned against delaying the project, saying the overcrowded rail network could stunt "growth across the entire nation".
One of the letter's signatories, London Chamber of Commerce and Industry (LCCI) chief executive Colin Stanbridge, said: “It is more important now than ever that not only does London continue to grow as global city, but that it is seen to do so.
“LCCI would like to see a firm commitment for Crossrail 2 which is vital for London, with its potential to secure 200,000 London based jobs, over 60,000 supply chain jobs across the UK, and tens of thousands of new homes."