The AFL says a High Court ruling preventing Optus from restarting a service allowing customers to watch matches on their mobile devices protects important commercial rights.
Friday's decision was a joint victory for the AFL and NRL, who were partners in the legal battle against Optus.
The AFL sold its five-year mobile and internet broadcast rights to Telstra for $153 million last year, while the NRL is still in negotiations over its mobile broadcast rights.
Friday's decision denied Optus the right to appeal a ruling against it made by the full bench of the Federal Court in April.
"The AFL welcomes the decision today to refuse Optus leave to appeal to the High Court," the AFL said in a statement.
"The High Court's decision ends this litigation, which Optus commenced against the AFL and NRL last year.
"The High Court's decision confirms the position taken by the AFL in relation to Optus' service and maintains the protection of important commercial rights."
Optus was ordered to pay the AFL's costs.
With the court process now exhausted, Optus will shut down its TV Now service, which was suspended indefinitely as a result of the April decision.
That decision overturned an earlier Federal Court ruling, which had allowed Optus customers to watch AFL and NRL games on mobile devices as soon as two minutes after they screened live on television.
Optus had argued it equated to customers recording shows for their own personal use, so did not breach copyright laws.
Despite Friday's ruling, Optus remained hopeful that the act, currently being reviewed by the Australian Law Reform Commission, would eventually be changed.
"The law as it stands imposes an arbitrary distinction between technologies," Optus' vice-president of corporate and regulatory affairs David Epstein said in a statement.
"Online storage is here and with us now so we think it's time to confront the realities of that. We can't shut ourselves off from the world."