Hannah Witheridge, 23, from Norfolk, and Leeds University student David Miller, 24, were found dead on the island of Koh Tao last September.
Burmese migrants Zaw Lin and Wai Phyo are on trial for murder after denying their involvement.
Andy Hall, a British migrant rights activist working with the defence team, said the development was the first breakthrough in the men’s case.
Dr Porntip Rojanasunand, from Thailand’s Central Institute for Forensic Science, told the court on Koh Samui that a garden hoe alleged to have been used in the killings carried the DNA of two men but there was no trace of either defendant.
She also raised concerns about flaws in the early investigations, including how forensic evidence was collected.
Mr Hall said: “The police previously said they did not test the weapon, so we asked for a retest.
“We recently got the results and the doctor came to court to testify that there was no trace of the two defendants.
“It is our case that there has never been any evidence to link the two boys to the murder weapon and our case over the coming weeks will focus on the reliability of forensic evidence.”
The bodies of the pair were found on a beach in September 15 last year.
Miss Witheridge had been raped while Mr Miller, who was from Jersey, died after being hit over the head before drowning in the sea.
Post-mortem examinations showed that both had suffered severe head wounds.
Both defendants, aged 22, initially confessed to the killings but later retracted these statements, saying they had been tortured.
Prosecutors say the DNA evidence, collected from cigarette butts, a condom and the bodies of the victims, links the two men to the killings and defence lawyers have been requesting that evidence for re-examination since April.