Prince Harry appeared to wipe away tears as he and his bride, along with the 600 guests packed into St George's Chapel, sang the last bars of Guide me, O Thou Great Redeemer.
The hymn was his mother's favourite and the 33-year-old chose to include it as part of the ceremony as a tribute to Princess Diana, 21 years after it was sung at her funeral.
Prince Harry, who looked nervous as he walked into the chapel with his brother and best man Prince William, appeared to struggle with his emotions as his stunning bride walked up the aisle on her own - with her father too ill to attend.
But the new Duke of Sussex was again overcome with emotion as the touching hymn was sung.
The song was performed at Diana's funeral at Westminster Abbey in 1997, when Prince Harry was just 12 years old.
Last year Harry admitted in a TV documentary he had only cried twice about his mother's death in a car crash in Paris, an event which shocked the world.
He said: "The first time I cried was at the funeral on the island," referring to Diana's private burial at Althorp.
"And only since then, maybe once more. There's a lot of grief that still needs to be... to be let out."
There were touching references to Harry's late mother throughout the ceremony.
Meghan carried a bouquet containing forget-me-nots, specifically chosen as they were Diana's favourite flower.
A spokesperson for Kensington Palace said: "The couple specifically chose them to be included in Ms Markle's bouquet to honour the memory of the late Princess on this special day."
Diana's sister Lady Jane Fellowes gave the only reading of the service, the eighth chapter of the Song of Solomon: “My beloved speaks and says to me, ‘Arise, my love, my fair one, and come away; for now the winter is past, the rain is over and gone’.”
She was joined at the service by Diana’s other sister, Lady Sarah McCorquodale, and her brother Earl Spencer, along with Harry’s Spencer family cousins.
Also present was former Prime Minister John Major, who was appointed special guardian to Princes Harry and William, responsible for legal and administrative matters, when Diana died in the Paris underpass.
Sir Elton John, Diana's close friend, was asked by Harry to play a medley of best-loved hits during the couple's lunchtime reception.
Meghan's jewellery also included nods to Diana, with her engagement ring - which Harry designed - featuring two stones from Diana's personal collection.
For the evening do at Frogmore House the Duchess of Sussex wore Diana's aquamarine ring.