A highly sensitive spectrophotometric method for ethyl carbamate (EC) determination was established through glutamate dehydrogenase/urethanase cascade reactions and the corresponding change in NADH concentration. The absorbance at 340 nm is linearly related to the EC concentration within the range of 0.3–50 μM, with a low detection limit of 0.00928 μM. The assay was further applied to analyse EC in mimic Chinese rice wine samples.