But Grigory Logvinov says the two people being expelled are career diplomats listed on the embassy website.
"They are absolutely legal, career diplomats... without any grounds called spies," Mr Logvinov told reporters in a sprawling press conference at the Russian embassy.
When asked how many Russian spies were left in Australia, Mr Logvinov replied: "Zero minus zero is still zero."
The ambassador also accused local spies of bad behaviour towards Russian diplomats in Australia, but refused to provide details.
"We have a bad history of improper behaviour of Australian authorities ... it happened in 2016, it happened in 2017," he said.
"There were a couple of cases, but once again I'm not going to elaborate, we agreed not to make any noise of it."
Foreign Minister Julie Bishop will meet with Mr Logvinov on Wednesday afternoon and will tell him the use of a nerve agent on foreign soil was unacceptable.
"In relation to a number of the statements that he has made, we do take issue with them," she told reporters.
"I haven't seen precisely what he's said but some of the comments that have already been put to me of course we reject."
Ms Bishop said the UK had provided Australia with a compelling case that either Russia was behind the attempted assassination or had lost control of its illegal stockpiles of nerve agent.
Mr Logvinov said Britain had provided no evidence Russia was behind the attack on the Skripals.
"We have seen this script before. This is standard Russian propaganda, we've seen it in relation to MH17, we've seen it over many years," Ms Bishop said.
Asked whether Australia would be pulling out of July's World Cup in Russia, Ms Bishop said a boycott is not being considered.
"The team, of course, will go," she said.
"When I was asked about it yesterday I was referring to the fact that Britain, for example, pursued an option of announcing that the royal family would not be attending the World Cup."
Mr Logvinov said President Vladimir Putin was waiting on recommendations on how to respond to the expulsion of more than 100 Russian diplomats from countries around the world including Australia.