Mr O'Flynn's comments come after reports that the Lib Dems, as part of any post-election coalition deal, would only give their support to a poll if they had a say on the wording of the question, the timing of the ballot, and who could take part.
But at a press conference in London on Wednesday, UKIP party spokeswoman Suzanne Evans said school children had been exposed to pro-EU propaganda and should not take part.
"It's everything there from colouring-in books on the Common Agricultural Policy for primary school children right up to research projects at university level.
"The amount of money that the EU is putting into this propaganda and throughout the entire education system is enormous," she said.
Mr O'Flynn added: "Let's be frank about it, opinion research shows that 16 and 17-year-olds are much more likely to be in support of Britain's membership of the European Union than is the population in general.
"And the electoral franchise does not allow 16 and 17-year-olds to vote and it's no wonder the Liberal Democrats wish to make this change but it's a gerrymandering change which is clearly intended to skew the result."
He also said there was a "vested interest potentially" in how EU citizens living in the UK would vote, if they were allowed to participate.
Asked if he thought school children were being brainwashed, Mr O'Flynn said: "It is an emotive term but I wouldn't demur from the description of brainwashing."
He called for a "fair" referendum which sticks to the current voting rules for UK general elections.
Commenting on the letter by 100 business leaders publicly backing another Conservative-led government, Mr O'Flynn said it was "no surprise" big business wanted to maintain the "status quo".
He said he accepted big business was unlikely to support UKIP because of its stance on the EU, but insisted the party had the support of small businesses.
Ms Evans indicated that UKIP would set out further details of its plans to cut business rates in its general election manifesto.
She also told the gathered media that the manifesto would not include a target number for UK immigration, saying too many politicians had talked about numbers and targets that could not be met.
The party previously had to deny a U-turn on a target after its spokesman Steven Woolfe said immigration would be capped at 50,000 per year.
On Tuesday, party leader Nigel Farage said he would like immigration to return to "normal" levels, which he said was about 30,000 a year.