Reports emerged on Friday the minor party's leader paid $900 a night to stay at the five-star Conrad London St James hotel during her trip to Europe in July.
In a post on Facebook, Senator Hanson sought to clarify claims made by the Sydney Morning Herald including suggestions the room was left empty for five nights while she flew to Italy.
"I can confirm that while I was in Italy, accommodation in London was cancelled," she said.
Senator Hanson embarked on a 10-day overseas fact-finding trip that included a sit-down interview with Seven's Spotlight program.
She said the broadcaster initially agreed to pay all travel costs but pulled out, leaving One Nation to foot the bill.
The allegations come after One Nation's Fire the Liar campaign raised $5 million from thousands of small donations over a few months.
The funds raised will pay for billboards and broadcast advertising to "reach Australians that Albanese ignores" at the next election, according to the One Nation website.
The statement on Facebook said these funds had been "quarantined".
One Nation senator Sean Bell dismissed criticism of the trip earlier on Friday.
"I can assure people ... when we go overseas, we do so making sure people get bang for their buck," he told News24.
Senator Bell assured donors money raised through the grassroots campaign had been "cordoned off" and could not be used for other causes, including international travel.
"All the money that was sent to that is being used on that issue," he said.
Senator Hanson has previously defended the overseas trip, which included a five-night stay at an Italian resort with mining magnate Gina Rinehart, insisting taxpayers had not paid a cent.
One Nation has faced increased scrutiny as it repeatedly out-polls the major parties.
Immigration Minister Tony Burke's last-minute decision to pull out of a National Press Club address has been claimed as a win for the minor party.
Mr Burke was expected to outline a new approach to immigration standards to meet the government's net migration target of 225,000 people.
Senator Bell said he welcomed the major parties coming to the debate.
"I'm glad we can all agree that One Nation was right about the need to end mass migration. Everyone agrees," he said.
"Now we're just arguing about the best way to do it."
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