An alpaca called Geronimo, who has twice tested positive for bovine tuberculosis, will be killed, the High Court decided.
The pet owner begs to be rescued, believing the tests return false positives.
Helen Macdonald was denied permission to test the animal a third time – but the court concluded with no “prospect” success in her bid to overturn an earlier decision.
The Department for Environment, Food and Rural Affairs (Defra) said he did not have to destroy Geronimo now.
Ms Macdonald lost her final appeal to the High Court this month and an order was issued for the alpaca to be modified.
More than 130,000 people have signed a petition calling on the prime minister to stop the killings, which have caused a public outcry.
Ms Macdonald, who owns a farm in Wickwar near Bristol, imported Geronimo from New Zealand in 2017.
An application for an order to stop the destruction order was considered by the High Court yesterday-but the judge said he needed more information from Ms Macdonald and from government lawyers before he could decide.
The hearing was adjourned until now, when Mrs Justice Stacey refused the application.
Geronimo first tested positive for bovine tuberculosis in September 2017 and he has been isolated ever since, according to the owner’s attorneys.
Catrin McGahey QC said nine other animals were subjected to the same testing regime as Geronimo showed no signs of illness after they were killed, indicating a potential inaccuracy in the tests.
He argued that Defra knew about it and did not disclose the information, which emerged as a result of a recent investigation in the Daily Mail.
Mrs Justice Stacey said the farmer’s complaint about non-disclosure was an “undesirable and backdoor way of finding a further route to appeal” and did not provide a contentious case.
The law firm representing Ms Macdonald said they were “extremely disappointed” and “surprised” at the outcome.
Dr Iain McGill, a vet advising Geronimo’s owner, said the decision made in the High Court was “unbelievable”.
He said Defra had “not produced any evidence” to back up claims that the tests used on Geronimo were more than 99% accurate.
“Geronimo’s diagnosis is more than unsafe,” he said. “It’s an abortion of justice.”
Ned Westaway, who represents Defra executive’s Animal and Plant Health Agency, said Ms Macdonald would be given the opportunity to make her own arrangements for Geronimo’s destruction.
The government insisted the test results and the options for Geronimo were carefully considered.
A Defra spokesperson said: “Bovine tuberculosis is one of the greatest animal health threats we face today and is causing devastation and distress for farming families and rural communities across the country, while costing the taxpayer to amounting to £ 100 million per year.
“Therefore, even if no one wants to save infected animals, we must do everything to solve this disease to stop its spread and protect the livelihood of those affected.”