The decision was taken at the league's AGM after a business plan put forward by the consortium negotiating a potential takeover failed to convince officials.
Gretna, whose entire staff was made redundant by administrator's days after their debut Scottish Premier League season ended, will be expelled from the league if a buyout is not completed after the weekend.
The club's spectacular fall from the top flight to the bottom tier means promotion for the SFL's two losing play-off finalists - Airdrie move into the First Division and Stranraer leap to the Second.
David Longmuir, the SFL chief executive, said: "Gretna were admitted to the Scottish Football League last Friday and today we have decided to invoke a rule to relegate Gretna to the Third Division.
"We have done this in consultation with the administrator and we have had a very good working relationship with him. He has agreed with the decision.
"A lot of it was based on, first of all, the administrator telling us that he did not see a sustainable business plan from the third party trying to buy Gretna.
"We also looked at the business plan and we agreed. We found many fundamental flaws with the business plan.
"We also have to recognise that there has been no work done on Raydale since 2006.
"There were a number of other issues around the areas of football debt, and UEFA and SFA rules on football debt, that the third party was unable to quantify and therefore give total clarity on how he was going to handle those debts.
"Therefore, we decided the risks were far too high."
The league will invite applications for a new club to join the SFL if a takeover is not completed by the "very early point of next week".