The store at 56 Liverpool Road is closing and the branch reopening at 78 Liverpool Road in the former Royal Bank of Scotland building.
Franchisee Ross Fairbairn bought the former bank in April and has been refurbishing the building a restaurant area on the ground floor and while first floor has been converted into a residential apartment.
The new Subway will also have its own customer parking to the rear.
It opens on Saturday, November 30 at 7am.
Mr Fairbairn said: "We look forward to serving our old and new customers and welcoming the community of Penwortham to our new store."