Sir Tom loaned the museum his medals, an FA Cup Final shirt, his Footballer of the Year trophy and his CBE and knighthood medals.
Now he has penned a letter to the Mayor of Preston, warning that he will reclaim all the items if the museum moves to Urbis.
'Keep soccer museum in Preston' - Sir Bobby
He said in the letter: "There seems no logical reason why thousands and thousands of football treasures should be transferred at enormous cost to a sterile building in Manchester when this money could be used support the National Football Museum.
"I have already informed the people concerned that my personal memorabilia currently on display in the museum will not be allowed to leave Preston."
The M.E.N. revealed in September that Manchester is set to become the museum's new home early next year due to a lack of funding for the site in Preston.
Under the plans, the museum's collections would stay in Preston but front-of-house exhibitions are set to be transferred to Urbis.
Sir Tom, 87, made 472 appearances for Preston North End and has 76 caps for England.
Bosses at the museum said Sir Tom's wishes would be respected.
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Henrys Dog (01/10/2009 at 13:13)
What better place for the museum than the worlds greatest footballing city.