Bolton, who called for the meeting after being given a vote of no confidence by party chiefs in January, lost a ballot of members with 63% backing his sacking.
The former soldier was hoping he could win the support of Ukip members at the meeting in Birmingham, but alienated much of the crowd with a threat to sue the party if he lost during his final speech on Saturday afternoon.
In an earlier address to the around 1,400 members, Bolton claimed his opponents were “the enemy within” the party.
But 876 of the 1,406 votes cast were in favour of removing Bolton.
Party veteran Gerard Batten, a founder of Ukip, was announced as the interim leader, while a new leadership election will be held within 90 days.
Batten has previously argued that Muslims should sign a special code of conduct rejecting violence.
When asked whether he intends to take Ukip in the direction of being more anti-Islam, he told a press conference on Saturday: “I’ve never made comments about Muslims, I’ve always talked about ideology and the literalist interpretation of that ideology.
“That is a big problem that is facing not just the UK but Europe and the Western world. I will be looking at policies of how we can address that.
“It’s all very well to talk about integration but one of the practical things we should do – our Government should do – straight away, is to stop the foreign funding of mosques and imans by places like Saudi Arabia and Pakistan who have exported extremist ideology to our country and other countries.”
Batten said he has not decided whether or not to stand for the leadership, added: “I honestly don’t know the answer to that. I’ll wait and see how it goes or otherwise over the next few weeks.”
Speaking after the result was announced, Bolton did not rule out quitting Ukip and forming his own party.
When asked if he would stay in the party, Bolton said: “Let’s decide that in a couple of days.”
Bolton’s position became increasingly precarious after it emerged he had left his wife for Ukip activist Jo Marney – a move which some in the party felt undermined his ‘family man’ image.
Leaked texts from Marney which contained racist and derogatory comments about Prince Harry’s fiancée Meghan Markle only added to the pressure on Bolton, who then broke up with the glamour model in order to stay on as leader and fight for his position.
Since then, he was spotted by HuffPost UK having an intimate dinner with Marney at an exclusive members-only club on the banks of the Thames.
He initially claimed the “romantic part” of the relationship with Marney is over, but repeatedly refused to rule out continuing his romance.
Ukip now faces its fourth leadership election since the EU referendum in June 2016.