Mr Obama met fellow Nobel laureate Suu Kyi at her lakeside villa in Myanmar's commercial capital Yangon, after arriving from the capital Naypyidaw where he discussed the nation's troubled reform process with President Thein Sein.
Ms Suu Kyi's National League for Democracy (NLD) party is widely expected to sweep polls in late 2015, but its figurehead is banned from the presidency by a constitutional clause.
Mr Obama and Ms Suu Kyi are due to hold a press conference after their talks in the garden of her villa in a reprise of their landmark meeting in 2012 which saw the US leader throw his political might behind Myanmar's transition from junta rule.
After talks with his counterpart Thein Sein late on Thursday, Mr Obama expressed cautious optimism hope for the once-cloistered nation that balanced out earlier warnings on the risks of "backsliding" on the transition.
"The democratic process in Myanmar is real," Mr Obama said.
"We recognise change is hard and you do not always move in a straight line but I'm optimistic."
During his two-night trip to Myanmar the US leader has also raised alarm over the direction of reforms, however, citing the cramping of freedom of expression, ongoing conflicts and the treatment of Myanmar's minority groups -- especially the Muslim Rohingya.
Mr Obama was whisked from Yangon airport to tour the British colonial-era secretariat building in downtown Yangon where Ms Suu Kyi's father, independence hero General Aung San, was gunned down by political rivals in 1947.
Their talks will be held at Ms Suu Kyi's lakeside family home, almost four years to the day after she was released from years of house arrest by the generals for her freedom struggle.
Her street, which also houses the US Embassy, was sealed off Friday with dozens of police at each end as well as a scrum of reporters and cameramen and some NLD members.
Most political prisoners have been released and elections have seen Ms Suu Kyi become a lawmaker, while foreign investors have arrived in lockstep with the lifting of most sanctions.
But the atmosphere has slowly soured, with many observers saying reforms have stalled.
Ms Suu Kyi cautioned against US "over-optimism" ahead of Mr Obama's visit, with even her star power earned as the torch-bearer of democracy during the dark junta years having waned in the eyes of some.
For his part, Mr Obama has been battered domestically with poor approval numbers compounded by a thumping defeat for his Democrats in last week's mid-term elections.
He has invested a large amount of political capital in Myanmar's transition from military rule and hopes his second visit will boost the process as elections edge closer.
His visit has coincided with the start of a debate on constitutional reform, in particular over the clauses effectively blocking a presidential bid by Ms Suu Kyi and reserving 25 percent of seats for the military.
While Mr Obama is cautiously optimistic on the long game for impoverished Myanmar, many ordinary people are not as easily convinced.
"I wondered when Obama first came, whether things will be better," 52-year-old street stall holder Minny Oo Aung told AFP in Yangon, where security is high, with clusters of police about every hundred metres.
"But there has been no improvement in our society or economy."