AbstractAn amperometric biosensor for ethyl carbamate (EC) was developed for the first time through the cascade reactions of urethanase and glutamate dehydrogenase (GLDH). Urethanase decomposes ethyl carbamate to produce ammonia, which converts to L‐glutamate under the catalysis of GLDH in the presence of α‐ketoglutarate and NADH. Then the change of NADH can be detected chronoamperometrically. The two enzymes were entrapped into chitosan/gelatine/γ‐glycidoxy propyl trimethoxy silane sol‐gel and immobilized on the surface of pyrolytic graphite electrode (PGE). The modified electrode was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Under the optimized conditions, the amperometric EC biosensor exhibits a linear detection range from 0.5 to 40 μM with a low detection limit of 5.30 nM. The biosensor was successfully used to detect EC in mimic Chinese rice wine samples, and satisfactory recovery and relative standard deviation were achieved.