Toronto failed to convince Super League clubs they had an adequate business plan, says executive chairman Robert Elstone.
The Canadian club withdrew from their inaugural season in the top flight in July, with owner David Argyle citing financial problems caused by the coronavirus pandemic, but expressed the hope that they would return next year under new ownership.
However, a meeting of the Super League board on Monday rejected their submission by a vote of 8-4 with one abstention.
Super League executive chairman Elstone said clubs were concerned by "the aggressive revenue targets on which the financial forecasts are based" and an independent committee concluded that a Canadian team would not be beneficial to the league.
Elstone said: "The board accepts that the decision to reject Toronto Wolfpack's application will divide the game's fans but, on the evidence presented to us, it would not be right for the development of the competition for Super League to accommodate a team in Canada in 2021.
"Every opportunity has been given to Toronto Wolfpack to provide the assurances our clubs need.
"However, our review of the club's recent submission identified a number of areas of concern, particularly regarding the aggressive revenue targets on which the financial forecasts are based.
"As part of our comprehensive investigation into this whole subject, Super League appointed an independent committee of sports industry experts, with representation from the RFL, to examine commercial opportunities for rugby league in Canada.
"Its findings were unanimous – that operating a team in a fiercely competitive North American sports market was non-strategic and added no material incremental revenue to Super League in the short or medium term.
"Separately, it was also apparent that no assessment of the scale and accessibility of the commercial growth that might accrue to the sport from entering the Canadian market was ever completed prior to the club's first entry into the sport."
Elstone broke the news to Wolfpack chairman and chief executive Bob Hunter, who said: "We're naturally disappointed. I thought we put a pretty good submission together.
"I think at the end of the day, there were a few different questions that probably didn't get answered, so disappointed but understood."
Toronto were formed in 2016 by Canadian entrepreneur Eric Perez and, after starting out in League 1, won promotion twice in three years to claim their place in Super League.
They played in front of crowds of 9,000 in their Championship season but hopes of capitalising on the signing of Sonny Bill Williams for 2020 were scuppered by an inability to play any games in Toronto.
Toronto businessman Carlo LiVolsi, the club's prospective new owner who had made a pledge to pay five months of unpaid wages to the players, made an online presentation to the club representatives which failed to win over the waverers.
LiVolsi had said in advance of the meeting that it was "Super League or bust" and Hunter confirmed the prospective new owner had no interest in rejoining either League 1 or the Championship.
Asked if there was any option to appeal against the decision, Hunter said: "I don't think so.
"The only person who could decide at this point in time would be David (Argyle) and I just don't think that David right now still may have the passion or the resources to take it.
"I'll be speaking to him shortly.
"We could certainly entertain a new owner but we're definitely out of Super League in 2021.
"Whether we re-apply to the RFL going forward, I can't really say, it's just too early."
Leeds were one of three clubs to support the Wolfpack case, along with the Rugby Football League, whose single vote was cancelled out by that of Super League's executive chairman Robert Elstone, a known sceptic of the Canadian club.
Super League must now decide whether to run with 11 clubs in 2021 or find a 12th member and its board will reconvene on Tuesday to consider its next move.
There would be no shortage of candidates if Super League goes for the second option, with Featherstone and London Broncos previously offering to take Toronto's place in 2020.
Toulouse, York and Leigh are other Championship clubs with Super League aspirations while Bradford could also come under consideration.