The Belgian drove for Virgin as a rookie last year and in general gave a good account of himself alongside the experienced Timo Glock.
However, he was still discarded shortly after the final race of the season in Brazil.
The 26-year-old has now finally resolved his future and will serve as understudy to Kimi Raikkonen and Romain Grosjean, attending all races this year.
D'Ambrosio, whose management representatives Gravity Sport Management are owned by Genii Capital, which also owns the Lotus team, said: "It's a great feeling to be joining Lotus. It's an exciting next step in my career.
"Now I have the opportunity to work with one of the top teams with great potential for my future development."
Team principal Eric Boullier added: "In Jerome we have someone we know well who integrates beneficially with the squad.
"He impressed as reserve driver for us in 2010, and ... also showed strongly in his rookie season as an F1 race driver."