Tests on the other 24 horses on the J4S Equine Nursery near Rockhampton came back clear, said Biosecurity Queensland acting Chief Veterinary Officer Rick Symons.
An anglo-arab filly died on the property last Saturday after contracting Hendra.
The horse who is to be re-tested was one of the four tested yesterday when they were showing slightly elevated temperatures.
"Those same-day test results came back clear so we are surprised by today's positive result and we will be re-testing to exclude a false positive.
"In the meantime this horse and any in-contact horses will remain isolated on the quarantined property at Cawarral.
"The biosecurity officers who are attending these horses each day continue to wear appropriate, high level Personal Protection Equipment for the duration of the response."
Dr Symons said today's results were for PCR testing, which showed any presence of the virus in the horses' blood or mucus.
"All of the nasal swab mucus samples were clear but the blood sample from this single horse shows signs of the virus," he said.
"We expect to receive serological test results early next week, which will tell us if any of the horses has had a reaction to the virus and is producing anti-bodies.
"Tests on the horse that died at Cawarral on Friday, August 7, the day before the already known Hendra horse died on Saturday, August 8, have confirmed it had also been infected with Hendra virus. Biosecurity Queensland has always treated this horse as a suspect case and this does not affect the response.
"The Cawarral property and a neighbouring property will remain under quarantine until such time as the site can be completely cleared of any further infection."
Blood tests on 18 people potentially exposed to the deadly virus have come back negative, Queensland Health has said this morning.
But follow-up testing will be needed in the next few weeks to clear them of having the virus which has an incubation period in humans of five to 16 days.
Four people who had significant exposure to the horse who died last Saturday are in the Rockhampton Hospital having five days of intravenous treatment with the anti-viral drug, ribavirin.
An unnamed person who was exposed to Hendra during the Redlands outbreak last year was treated with ribavirin and never developed the deadly infection.
Princess Alexandra Hospital infectious disease physician Geoffrey Playford said ribavirin had been used since the early 1970s, most commonly in the treatment of patients with viral hepatitis who can take it for between six and 12 months.
"The most common side effect that we see is an anaemia, the destruction of red blood cells, which tends to be self-limiting upon cessation of the drug," he said.
Men and women of child-bearing age who take ribavirin are advised to remain on contraceptives for six months after stopping use of the drug because of the potential for birth defects.