"How do you make a broken family whole?" she said.
"The same way you make a nation whole. With love and understanding — and with small acts of compassion. With bravery. With unwavering faith."
Americans, she said, were "coming together" despite Trump's divisiveness.
"We have shown that the heart of this nation still beats with kindness and courage," she added. "That's the soul of America Joe Biden is fighting for now."
The president faced a barrage of criticism on opening night too, notably from former first lady Michelle Obama who said Trump lacks the character and skills for the job.
"I thought it was a very divisive speech, extremely divisive," Trump told reporters at the White House.
In her pre-taped remarks to the convention, Obama urged Americans to rally behind Biden, her husband's former vice president, in the November 3 presidential election.
"Donald Trump is the wrong president for our country," Barack Obama's wife said in unprecedented criticism of a sitting US president by a former first lady.
Later Tuesday on a visit to battleground state Arizona, Trump knocked Obama for delivering taped remarks.