The singer announced last month, after weeks of speculation, that he was teaming up with businessman Ryan and their takeover was expected to be formalised on Friday.
But efforts to raise enough cash for the deal - through the Crowdfunder.co.uk website - have faltered with only £757,796 of the £2million target raised by Thursday's deadline from 514 backers.
A statement issued on Thursday by the Tomlinson Ryan Trust, established by the pair, said the failure to reach their target meant they were unable to meet the Football League's 'fit and proper person' test for club ownership.
Tomlinson has said he had been "misled" but hopes to stay involved, while Ryan, who last November stepped down as club chairman, confirmed the joint venture was over and that he was finished with football for good.
Ryan told Press Association Sport: "When Louis says he was misled, he's certainly not referring to me, I spoke to him on Thursday.
"I don't want to go into the ins and outs. As far as I'm concerned the whole thing is finished.
"I'm finished with football. I just want to live my life a different way now.
"I hope Louis does stay involved because he's a great supporter of the club and I know he will do all he can.
"But the reality is, in my opinion, it's over. Maybe down the line, certainly where Louis is concerned, he will be able to get involved again.
"But our partnership hasn't worked out. The crowd-funding was an absolute failure, but it's been a combination of that and the Football League's stance.
"I don't want to go into into details. I don't want to go there."
Lifelong fan Tomlinson and Ryan hosted a press conference last month outlining their plans for the takeover and launching the crowd-funding bid.
Those who have pledged cash will not lose out as they do not have to pay up if a project does not meet its target under Crowdfunder's rules.
A spokesman for the investment site said: "Crowdfunder is an all-or-nothing model. If a project doesn't reach its target, no money is taken from backers."
The Trust's statement on Thursday said the takeover was in "serious doubt", although it had previously stated that would take place irrespective of the success or failure of its crowd-funding project.
Ryan is understood to have met with Football League representatives this week in a bid to reassure them he and Tomlinson had the finances to run the club.
He added: "I've given my all for 20 years, including about £10million, and although I wouldn't say I feel embittered I've had enough.
"I've got two daughters and they're both telling me I look ill. They've said they want a dad, not an owner of a football club anymore.
"I've decided not to be an owner. I will walk away completely from football - there's a lot of things I don't like about it.
"But I wish Doncaster well and I hope they do really well next season."
The Football League issued a statement on Thursday night saying it had yet to receive an acceptable business plan from Ryan and Tomlinson.