Knights are currently top of the Championship with two games of their season remaining.
But even if they win the title they will not be promoted after the RFU ruled that the capacity of their Castle Park home is too small for the Premiership.
The governing body announced on Tuesday that Knights and title rivals Ealing Trailfinders - the only two second tier sides to have applied for promotion - both failed to meet minimum standards criteria.
“The independent audit has found that neither club has successfully met the minimum standards criteria based on capacity (as well as other factors),” said the RFU’s statement.
Premiership stadia must accommodate more than 10,000 fans. Castle Park’s current capacity is 5,183.
Knights responded today by saying they were “disappointed, dismayed and disheartened” by the RFU’s decision and plan to launch an appeal.
The club believe they can increase Castle Park’s capacity in time for the new season – through temporary seating – and say ‘a stand-by ground is now available should unexpected delays occur’.
Knights said in a statement: "Doncaster RFC are disappointed, dismayed and disheartened by the decision of the Rugby Football Union Board to disallow the club’s application to play in the Premiership league should the Doncaster Knights win the current season’s Championship league contest. The Club will be appealing the decision.
“Within the audit process, the only failure by the Club is to, currently, not be able to demonstrate a capacity crowd of 10,001 at the Club’s ground, Castle Park. All other aspects of the audit, it seems are in order and Castle Park has, on a number of occasions, demonstrated its proficiency in hosting significant events via both Women’s and Under 20’s international matches.
“At this stage, it can only be stated that the DRFC Board have every confidence that the required capacity could be delivered for season 2022/23 with a stand-by ground now being available should unexpected delays occur.
“There has been significant debate about the Premiership being open to promotion and relegation, which can only be good for the aspirational nature of clubs, players and fans alike. The current Championship contest is approaching its climax with at least three teams statistically able to take the winner’s title. By the beginning of April the victor will be known.
“Excitement in the camps and amongst rugby supporters in general is high but that has now been dashed at a stroke via yesterday’s untimely RFU statement, producing a resurgence of subsequent outpourings of angry feelings towards the governing body at a time when harmony should prevail.
“Sensibility and fairness dictate that a delay to a decision being made could have been better for all concerned. Now, the teams play the final matches of the season in an atmosphere of despondence, albeit an administrative box has been ticked.”