Tutu placed his foot in a block of wet concrete, joining a list of Nobel laureates who had done so.
Other famous footprints outside the Tumulus building at the site included those of former president Thabo Mbeki and former UN secretary general Kofi Annan.
The left-handed Tutu quipped as he put his signature next to his footprint: “God made people perfect; the others He made right-handed.”
He asked: “Waar’s my kous?” (where is my sock) when he finished making his footprint.
The project represents support for the belief that the first humans walked out of Africa to populate the rest of the planet.
A jovial Tutu joked with journalists and tourists while he was taken on a short guided tour of the Cradle’s exhibition centre.
The 1984 Nobel Peace Prize laureate and former head of the South African Anglican Church described the brief ceremony of leaving his footprint as “humbling” and “a huge privilege”.
Tutu burst out laughing while telling a story of how he and his wife were in the Netherlands when a 400-year-old school was named after him.
“A little girl rushed up to me and asked me if I was there when the school started,” he said.
“It is scientifically proven that we belong to one family. That is why ubuntu is so important. Ubuntu started here.”
Tutu’s footprints joined the human database at the Cradle of Humankind, which is also famous as the location where a pre-human skull, now known as Mrs Ples, was discovered. A complete hominid skeleton, called Little Foot, was also unearthed at the site.