Authorities are still determining how many people had contact with the latest infected horse, so that monitoring of their health, and blood tests, can be arranged.
In all, 17 people are already being monitoring, but no-one is considered to have had more than light to moderate exposure to the virus, which is carried by native flying fox bats.
The virus can be passed on to horses and in seven known cases since its discovery in 1994, can infected humans. Four of those infected died.
Those exposed undergo initial blood tests, with follow-ups three weeks, and six weeks, later.
The first reported case was at Kerry, near Beaudesert, in Queensland, followed by the case near Wollongbar, on New South Wales's North Coast.
Today, Biosecurity Queensland said it was managing a fresh case at Mt Alford, in the Boonah area.
Queensland chief veterinary officer Dr Rick Symons said a horse at a local property had been sick on June 20 and was euthanised.
"A second horse became sick on 1 July and samples from this horse were sent to Biosecurity Queensland for testing.
"Test results overnight have confirmed this as a case of Hendra virus infection.
"The sick horse was euthanised. There are eight other horses on the property that we are monitoring closely.
"Tracing is under way to determine if the deceased horses came into contact with any other horses at adjoining or nearby properties.
"Queensland Health, key horse industry groups and the Australian Veterinary Association have been notified of this latest case."
Symons said Biosecurity Queensland staff will attend the Mt Alford area to provide information to neighbours of the property concerned.
This property is being quarantined to restrict the movement of horses on and off it.
The remaining eight horses will be monitored and tested.
Once officers from Biosecurity Queensland and Queensland Health have been able to speak with those people that treated the horse, a determination of whether anyone may have been exposed can be made.
There are no links between the horses or the properties in the two incidents.
Dr Symons said a third property had been quarantined as part as the Beaudesert incident.
This was a precautionary measure after tracing of horses on the original infected property at Kerry showed a possible contact with a horse at the third property.
"We have also received the first test results on the 20 horses at the second Beaudesert property at Biddaddaba," he said.
"They were negative. This is the first of three rounds of testing required to clear the property."
Symons repeated his advice that vets, horse owners and the community should be vigilant and report any suspected cases of Hendra virus to Biosecurity Queensland and most importantly, to take appropriate precautions when handling any sick horse.
"Fruit trees are flowering at the moment and there is a lot of flying fox activity. Horse owners are reminded that they should not place feed or water under trees where flying foxes gather," he said.