Corina Abraham appealed the granting of Aboriginal heritage approval for Roe 8, which is the most contentious part of the largely federal-funded near $2 billion Perth Freight Link project.
The road aims to divert heavy trucks onto a direct route to Fremantle Port and will be the state's first toll road. Supreme Court of Western Australia Justice Janine Pritchard dismissed the challenge on Wednesday.
Ms Abraham said she intended to fight on and would look at "all avenues".
"We're the custodians of this country and as a custodian, I will continue and endeavour this fight for my cultural rights and obligations as a Noongar person," she told reporters outside court.
Ms Abraham said she was disappointed with the decision.
"Barnett's government shows clearly a lack of respect in regards to the cultural significance we have as Aboriginal people to our country.
No one, not Barnett, no minister is going to come and tell me that we can't stop the destruction and erosion of our lands that are continuing throughout our state at a dramatic rate.
"Black, white or brindle, let's stand up together and face what Barnett's government is doing to this beautiful state."
She lashed the Department of Aboriginal Affairs for deregistering culturally significant sites, including the Beeliar Wetlands and others where bones and artefacts had been found.
Greens senator Scott Ludlam said it was another chapter in a shameful history of Indigenous dispossession in the state.
Ms Abraham was supported by Save Beeliar Wetlands convenor Kate Kelly, who said the decision was extremely disappointing but Perth Freight Link opponents would not give up.
The protest group - which says one-quarter of the wetlands will be cleared for the Roe 8 project - has raised more than $45,000 to fund its High Court challenge to the Environmental Protection Authority's approval of the project and also pay for previously incurred court costs.
The state did not seek costs from Ms Abraham.