Canberra officials said the sanctions target Russian national Sergey Dubinsky and Ukrainian Leonid Kharchenko, who were found guilty in absentia of murder and other charges by a Dutch court last year, after 298 passengers and crew died in the MH17 disaster.
Sergey Muchkaev, a colonel with the Russian Armed Forces, is also included in the sanctions by Australia.
He commanded the anti-aircraft brigade that supplied the weapons system which downed flight MH17, Canberra said.
There were 38 Australians on board when the Boeing 777 en route from Amsterdam to Kuala Lumpur went down over eastern Ukraine in July 2014.
"As either separatist leaders... at the time of the downing of Flight MH17, or a member of the Russian Armed Forces, the three people sanctioned today actively supported actions and policies that threatened the sovereignty and territorial integrity of Ukraine," said Australian Foreign Minister Penny Wong.
She added that the sanctions against Dubinsky, Kharchenko and Muchkaev demonstrated Australia's "ongoing commitment to hold to account those responsible for the downing of Flight MH17."
The third person convicted in The Hague last November, Russian Igor Girkin, had already been sanctioned by Australia in 2014 for supporting separatist activity in eastern Ukraine, Wong said.