Mr Macron and Ms Le Pen secured the two berths for the run-off in May 7 in a result that saw the two movements that have dominated French politics for 60 years eliminated in the first round, partial results showed.
Mr Macron, who quit current president Francois Hollande’s Socialists only last year ago to launch a new party, led the way with 23.7% of the first round vote, according to an exit poll by Ipsos and Sopra Steria.
He led his Front National challenger Ms Le Pen (21.7%) by 2%, with scandal-plagued Gaullist Francois Fillon and far-left challenger Jean-Luc Melenchon tied in third on 19.5%.
Socialist candidate Benoit Hamon trailed in a distant fifth out of 11 candidates with just 6.2% of the vote for the governing party.
Mr Macron, 39, thanked supporters, saying: “In a year we have changed the face of French politics.”
Thanking Mr Fillon and Mr Hamon for their early endorsements made during their concession speeches, he said: “Tonight I start to gather together the French people.
“I want to thank the millions of Frenchwomen and Frenchmen who voted for me.
“I am aware of the burden. It is a joy, but a serious joy, I feel tonight.”
Ms Le Pen, whose father Jean-Marie stunned voters by qualifying for the run-off in 2002 before losing heavily to incumbent Jacques Chirac, told voters: “You have the choice of an alternative, a true one.
“What I propose to you is a big alternative, the fundamental alternative that will put other faces in power.”
That appeared to be a jibe at Mr Macron, the former finance minister who was a protege of Mr Hollande before striking out on his own.
Aside from seven years from 1974 when Valery Giscard d’Estaing, the candidate of a party which nonetheless was allied to the Gaullists, was elected president, the run-off marks the first time since the Fifth Republic constitution was adopted in 1958 that both the Gaullists and Socialists have been rejected in the vote for the keys to the Elysee Palace and it is the first time neither movement has featured in the run-off.
Mr Fillon joined fellow conservative politicians and Socialist prime minister Bernard Cazeneuve in throwing their weight behind Mr Macron as an anti-Le Pen candidate in the second round of voting on May 7.
Mr Fillon, who had been favourite to win the presidency until he was embroiled in a “fake jobs” row concerning his Welsh wife Penelope, told reporters in France: “The voice of the right and centre can be heard in the (upcoming) parliamentary elections.
“While waiting, we have to choose: I don’t do it with joy in my heart but abstention is not in my genes.
“The Front National has a history known for its violence and intolerance: there is no other choice, I will vote in favour of Emmanuel Macron.”
Alain Juppe, the former prime minister and current mayor of Bordeaux, who was favourite to become president until he was beaten by Mr Fillon in a Gaullist primary before the Penelopegate scandal emerged, also backed Mr Macron “in his fight against the extreme right”.
Bookmakers made Mr Macron the odds-on favourite to win the run-off, with both Ladbrokes and Coral offering 1-6 and William Hill 1-8, with Ms Le Pen at 4-1, 7-2 and 9/2 respectively.
The last opinion polls before voting opened on Sunday showed Ms Le Pen and Mr Macron narrowly ahead of Mr Fillon and Mr Melenchon in what was billed as one of the most unpredictable elections in generations.
Pro-European Mr Macron was the Socialist finance minister until the autumn, when he quit to set up the En Marche! movement, which he defines as centrist, and which has attracted support from left, centre and right.
The anti-EU Ms Le Pen’s campaign majored on jobs, security and the threat from Islamic extremism.
It also saw her deny French state complicity rounding up Jews for the Nazis in the Second World War, but she also picked up muted plaudits from US President Donald Trump.
Speaking at the White House after a terrorist attack on Paris last week left a policeman dead, Mr Trump said she was “strongest on borders, and she’s the strongest on what’s been going on in France”.
Speaking after the polls were released Ms Le Pen told the public: “You have the choice of an alternative, a true one.
“What I propose to use is a large alternative, the fundamental alternative that will put other faces in power.”
Mr Melenchon said he would wait before the official results were in and did not instruct his supporters who to back on May 7.
The country is going to the polls to elect the successor to Mr Hollande, who is not running after serving a single term in office.
Earlier, thousands of French expatriates had queued for hours at polling stations in London to cast their votes.
There were already long queues around the block at the Lycee Francais Charles de Gaulle, near the Natural History Museum in Kensington, London, before the polling stations opened at 8am.
Polling stations have also been set up cities including Birmingham, Bristol, Belfast, Leeds, Manchester and Edinburgh for the large expatriate community to cast their votes, with 70 polling stations in total across the UK.