Southampton admitted spying on Middlesbrough before the first leg of the play-off semi-final, along with Oxford United at Christmas and Ipswich Town last month.
When Saints hosted Ipswich on 28 April, the two teams were in direct competition vying to finish second and earn automatic promotion. The game ended 2-2.
Southampton have no further right of appeal to the Court of Arbitration for Sport.
The Championship play-off final will now go ahead at Wembley on Saturday between Hull City and Middlesbrough (15:30 BST kick-off), with a place in the Premier League on the line.
The fixture is dubbed as the most lucrative game in world football, with promotion worth a minimum of £200m in broadcast revenue and parachute payments.
“A league arbitration panel has dismissed Southampton Football Club’s appeal against the independent disciplinary commission’s sanction following the admittance of multiple breaches of EFL regulations,” the EFL said on Wednesday evening.
Saints called the ruling “an extremely disappointing outcome”.
It added: “While we fully acknowledge the seriousness of this matter and the scrutiny that has followed, the club has consistently believed the original sporting sanction was disproportionate, a view that has been widely shared by many in the football community over the last 24 hours.”
Hull boss Sergej Jakirovic told a news conference before the final that his club had become “collateral damage” in the drama.
“We can say everything is unfair in this last two weeks. You don’t know what’s going on,” he told BBC Radio Humberside.
The Tigers’ owner, Acun Ilicali, told Sky Sports on Wednesday that he was unhappy that his club must face different opponents at short notice, and did not rule out legal action should they lose the final.
But Ilicali conceded that there was no other option but to play the fixture against Boro “in order to finish this mess”.