The London mayor misheard Ziad Al-Bandak and believed he was claiming to have family ties with the son of God.
Mr Al-Bandak eventually revealed he was joking to emphasise the deep-rooted ties of Christians in Jerusalem's sacred Old City, where the mayor has been on a visit.
The final day of his trade trip to Israel and Palestine, included heading to the Temple Mount/Haram al Sharif in Jerusalem's Old City.
The compound is the third-holiest site in Islam and the holiest site in Judaism. He visited the Dome of the Rock, the Western Wall and Holy Church of the Sepulchre.
Mr Johnson also observed a minute's silence to commemorate Armistice Day while overlooking Jerusalem at the Mount of Olives.
During the Old City tour, Mr Al-Bandak told the MP: "My grand, grand, grand, grandmother used to be the babysitter of our Lord Jesus Christ."
Mr Johnson replied: "I'm sure that's true. And Adam and Eve had a role in that - and Abraham."
Mr Al-Bandak replied: "Abraham was before that."
Mr Johnson pressed: "So you are related to Jesus?" The adviser to Mahmoud Abbas said: "Yes."
The Conservative MP added: "My God. Isn't there a book about that? The Da Vinci Code?"
Mr Al-Bandak replied: "No, the Da Vinci Code is very cynical."
Still appearing to be unsure about what he had heard, Mr Johnson asked: "So you're related to Jesus?" It was then clarified Mr Al-Bandak said babysitter.
Mr Johnson said: "I thought you said his baby sister.
"I was thinking Christ, because according to Luke he did have brothers and sisters."
Mr Al-Bandak, an adviser on Christian national and international relations, confirmed: "I'm not related."
The mayor joked there would have been considerable Hollywood interest in Mr Al-Bandak's story if he was related to Jesus. At this point, Mr Al-Bandak clarified he was joking.