A pyrrolidine-catalyzed three-component reaction of azlactone, N,O-acetal and alcohol for the synthesis of α,β-diamino ester was reported. The N,O-acetal served as the imine equivalent to occur Mannich reaction with azlactone, and the in situ generated α-functionalized azlactone subsequently underwent a ring-opening process in the presence of alcohol. A series of α,β-diamino esters were obtained in 36–99% yields under mild reaction conditions. The product could be synthesized on gram-scale and transformed into the α,β-diamino acid.