The union's written application for a judicial review on the introduction of fees for claims in Employment Tribunals and Employment Appeal Tribunals was rejected yesterday without a hearing.
Unison challenged the introduction of fees as it said it could prevent workers people from exercising their employment rights.
Monday will be a landmark date in employment law with the introduction of tribunal fees. This means employees must pay fees of £160 to lodge a claim plus £950 if the claim goes to an employment tribunal.
The Government is introducing the fees to reduce the number of employment tribunal claims being made.
Unison general secretary Dave Prentis said court's the decision is disappointing, but Unison would continue to challenge tribunal fees.
"The Government's plans to ration access to justice by introducing fees into Employment Tribunals and Employment Appeal Tribunals are unjust and discriminatory," Prentis said.
"We believe that that the Government should not put a price on justice and stop working people from exercising their employment rights.
"We are renewing our application and seeking an oral hearing when we will also be applying for a stay on the introduction of the new fees regime."
Next year, Acas will role out an early conciliation service, another measure designed to help employers and employees settle workplace disputes without the use of an employment tribunal.
The service, which is expected to be launched in April 2014, means anybody who wants to lodge an employment tribunal claim will have to notify Acas first and then have one month to resolve the dispute.
"At Acas, our advice is always that it is better to resolve disputes at the earliest possible stage, ideally in the workplace itself. Our training, good practice guidance and helpline all support resolution of problems," said Acas chief executive Ann Sharp.
"Early conciliation extends the part we play in avoiding formal tribunal hearings."