3,4-Dihydropyrimidin-2(1H)-ones were synthesized via α-chymotrypsin-catalyzed Biginelli reaction between aromatic aldehyde, urea and ethyl acetoacetate. Through optimization, the optimal reaction conditions were obtained as follows: the reaction medium was ethanol, the molar ratio of aromatic aldehyde to ethyl acetoacetate and urea was 1:1.5:1, the amount of α-chymotrypsin was 20 mg, the reaction temperature was 60℃ and the reaction time was 96 h. Under this optimum reaction conditions, a series of 3,4-dihydropyrimidin-2(1H)-ones derivatives having a yield of 55% to 63% were obtained. The method has the advantages of mild reaction conditions, simple operation and broad application range of the substrates.