In an infamous 1996 interview with 60 Minutes, the right-wing party's leader responded "I don't like it" while offering her thoughts on the annual LGBTQI festival.
"I don't like it because it's promoting something ... that is not natural," she said at the time.
Her "I don't like it" comment was co-opted by drag queen Pauline Pantsdown in 1998 and remixed into a parody dance song, which made it into the Triple J Hottest 100 list of that year.
Three decades later, Senator Hanson said she acknowledged the economic benefit of the pride parade, adding that it was "acceptable" Australia had legalised gay marriage.
"I still don't like the gay Mardi Gras, but that's my opinion," she told News24 on Sunday.
"Have I ever been to one? No. Do I intend to go? No, I don't.
"I think your own, personal sexuality should be kept ... behind closed doors."
Her comments follow revelations One Nation's candidate for WA's upper house at the 2025 election, Parminder Singh, made several anti-gay comments in a video on social media.
Mr Singh, who was third on One Nation's upper house ticket at the state election, has since been removed from the party's website, after the video became public.
During the week, One Nation MP Barnaby distanced the party from its former candidate's views, describing it as "one of the gayest parties in Australia".
Senator Hanson also rejected Ms Singh's comments, saying she never heard similar views from him when he was a candidate.
"That's his personal thoughts and not my thoughts whatsoever," she said.
The firebrand politician also defended her inability to name the leaders of crucial allies and regional powers when questioned about her foreign policy credentials as a would-be prime minister.
In an earlier interview with News24, the One Nation leader said diplomacy with Australia's neighbours would pose "no problem", despite her notoriously using her maiden speech in 1996 to declare the nation was "in danger of being swamped by Asians".
Asked if she could name the president of Indonesia or the prime minister of Japan, two major regional powers and key Australian trading partners, Senator Hanson said she couldn't.
On Sunday, she said she would learn the names when it was necessary.
"I still haven't looked up who the prime minister of Indonesia is because I don't need to know ... I'm not the foreign minister, I'm not the prime minister," Senator Hanson said.
The One Nation leader also reiterated her party's position that Australia's net migration target should be 130,000 a year - a significant cut on current levels.
She suggested the reduction could come at the expense of international students, a major source of income for universities, describing the intake system as a "farce" and a "scam" providing a back door into the nation.
But she was adamant any cut to migration should not alter backpacker levels.
"We cannot stop those people coming in; if they did, the farmers would go under," Senator Hanson said.
None of the major parties have outlined which visa categories should be cut to meet their reduced migration targets.
