The 99-year old, who is being cared for in a hospice, made a rare public appearance at the memorial at Emory Church in Atlanta.
He was president from 1977 to 1981 while his first lady Rosalynn, who died aged 96, undertook work to help a string of charities.
Carter, who is receiving hospice care in his home, was accompanied by a doctor for the service on Tuesday.
He was seated in the front row draped in a blanket with an image her face etched on it.
Jimmy and Rosalynn Carter were married for 77 years, the longest marriage of any first couple in US history, and both built a lasting legacy outside the White House through their humanitarian work.
Mrs Carter became a champion for mental health charities. Her husband, a former peanut farmer, became president when the US was in the grip of the Cold War.
Amy Carter, the first couple's only daughter, tearfully read a letter Jimmy Carter wrote to Rosalynn 75 years ago while he was serving in the Navy. Amy Carter, who was seated next to her father, was often seen holding his hand throughout the service.
"My mom spent most of her life in love with my dad. Their partnership and love story was a defining feature of her life," she said. "Because he isn't able to speak to you today, I am going to share some of his words about loving and missing her."
In the letter, Jimmy Carter wrote, "While I am away, I try to convince myself that you really are not, could not be as sweet and beautiful as I remember. But when I see you, I fall in love with you all over again. Does that seem strange to you? It doesn't to me."
A dozen political leaders were present at the the invitation-only tribute service for the former first lady.
President Joe Biden and first lady Jill Biden sat in the front row along with former President Bill Clinton and former first lady Hillary Clinton, as well as former first ladies Laura Bush, Michelle Obama and Melania Trump.
The memorial was one of a series of events to mark her life which will close with her burial in her hometown.