Traditional Owners appeared in the High Court Tuesday to make their case against the Singleton water licence.

The groundwater licence authorises Fortune Agribusiness to extract 40 gigalitres of groundwater at Singleton Station, south of Tennant Creek, every year for 30 years once it's running at full capacity, making it the largest groundwater licence issued in the Territory.

Traditional Owners noted in a statement on Tuesday morning that after 30 years, this volume of groundwater will equal draining Sydney Harbour twice.

After fighting for six years to protect sacred sites and their culture, native title holders are pleased to have their case heard in the High Court, the Central Land Council General Manager, Dr Josie Douglas, said on Tuesday afternoon.

"They have travelled two days from their homelands to be here," she said.

"It's an important opportunity to have their concerns about the Singleton water licence taken seriously."

Traditional Owner and 2026 Order of Australia medal recipient Frankie Holmes said: "If the water is gone, the plants are gone, the animals are gone, the sacred sites are gone."

"Water is our life blood. Water is life," he said.

Mpwerempwer Aboriginal Corporation director Dawn Swan said she felt "energised" listening to the case in court.

"It's really exciting, it's an eye opener. We hung in here for six years. We did it for our Elders and our future generation," she said.

"I'm going away spiritually motivated; it's making us stronger."

Six native title holders from the Mpwerempwer Aboriginal Corporation, supported by the Central Land Council, are in Canberra challenging the groundwater licence.