Ethyl carbamate (EC) is a widely recognized carcinogen present in fermented foods, and enzymatic degradation is a safe and effective strategy for its reduction. An EC-degrading esterase derived from Acinetobacter calcoaceticus (AcECH) was previously identified. However, volatile ester compounds in Chinese Baijiu compete with EC for enzyme binding, thereby affecting degradation efficiency and flavor quality. In this study, molecular docking, molecular dynamics (MD) simulations, enzyme kinetics, bio-layer interferometry (BLI), and gas chromatography–ion mobility spectrometry (GC-IMS) were employed to investigate the substrate specificity of AcECH. Based on computational prediction and experimental validation, F157G has been identified as the most suitable mutant for improving substrate recognition. Compared with the wild-type enzyme, the degradation rates of ethyl acetate, ethyl valerate, and ethyl hexanoate decreased to 64.23%, 49.28%, and 34.08%, respectively. Meanwhile, F157G showed enhanced apparent binding affinity toward EC while largely maintaining EC hydrolytic activity, and reduced the degradation of desirable volatile esters, indicating an altered substrate preference of AcECH.