After 25 hours of counting in the King's Hall in Belfast, Ulster Unionist Nicholson was confirmed on Tuesday evening as the third incumbent to book his ticket back to Brussels.
Sinn Fein's Martina Anderson emerged victorious after the first round of counting last night and Democratic Unionist Diane Dodds was declared elected earlier on Tuesday.
All three were loudly cheered by supporters as their successes in the 10 candidate race were announced.
Mrs Anderson, noting that Sinn Fein was on course to win four seats across the island of Ireland, hailed her party's performance.
"The voice of the voters have been heard throughout Ireland, they have endorsed the Sinn Fein message that there is a fair way," she said.
"The Sinn Fein result is part of a national story reflecting the growth of support for Sinn Fein's strategy for change. There will be a national Sinn Fein team of MEPs going to Europe who will put Ireland first - north, south, east and west."
Mrs Dodds said she would fight to secure the best deal for Northern Ireland in Europe.
"The DUP has always been a Eurosceptic party, we are not Johnny-come-latelys to Euroscepticism," she said.
"What we do believe is that this election really wasn't about whether we are in Europe or out of Europe. This election was really about who would go back to Europe to get the best deal for Northern Ireland, to work hard for Northern Ireland, to stand up for Northern Ireland and that's what I will be doing, as well as continuing my campaign for a referendum to give the British people their democratic say on our relationship with Europe."
Mr Nicholson has been a Euro MP for 25 years.
"I am the longest serving member ever elected in Northern Ireland," he said.
"I look forward to taking up the challenge because the next five years are going to be very challenging in Europe to get change and hopefully get a referendum."
He insisted the UUP was also in good shape on the back of his win and gains in the local government election.
"The party's health is the best it's been for many a long day," he added.
The count, which used the single transferable vote method, was suspended close to 1am today after almost 16 hours counting and resumed at 9am.
The result of the eighth and final stage of counting was announced at 6pm.
The SDLP's Alex Attwood was eliminated after the transfer of votes for the DUP's Diane Dodds.
TUV leader Jim Allister was eliminated at stage six this afternoon while Alliance's Anna Lo was excluded after stage five this morning.
Ulster Unionist leader Mike Nesbitt said Chief Electoral Officer Graham Shields had major questions to answer about the slow pace, adding that if he could not sort it out by 2015 then he should resign.
DUP MP Nigel Dodds blasted the process as a "shambles".
His wife Diane had still not be declared as elected last night while Jim Nicholson of the UUP and TUV leader Jim Allister faced a nervous wait to see who would take the third seat.
Northern Ireland will today be among the last constituencies in Europe to declare its full list of winning MEPs. Only the Balkans matched the snail's pace of the counting process seen at the King's Hall in Belfast yesterday.
The ballots arrived at the count centre already verified yesterday morning, a move which allowed counting to start at 9am.
However, the first result was not announced until 6.30pm. And by 1am this morning – when counting was halted until 9am – there was just one seat officially filled.
Graham Shields said the particular way the count developed could not have been reasonably foreseen.
Mr Shields said the episode underlined the need for electronic vote counting in Northern Ireland.
"The electronic counting process, in my opinion, is an absolute necessity for going forward because none of us want to be here for days on end," he said.
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