Enzyme degradation has emerged as the most promising method for controlling biogenic amines (BAs) in fermented foods due to its high specificity and minimal side effects. However, no studies have been reported on the enzymatic degradation of BAs in Douchi. Therefore, this study systematically investigated the effects of the Bacillus subtilis T2 multicopper oxidase (CotA) on the BAs, active substances, and flavor quality of commercially available Douchi. After CotA treatment, the BA contents of Chongqing Douchi (CQ) and Hubei Enshi Douchi (HBES) were decreased by 22.43% and 45.35%, respectively, while the total BA concentration of Sichuan Yibin Douchi (SCYB) was not significantly changed. Meanwhile, CotA treatment had no negative impact on the total phenolics and total flavonoids of these three Douchi. However, the CotA treatment had a significant effect on the flavor quality of these Douchi samples. Compared with the control group, CotA treatment increased the levels of multiple free amino acids in CQ and SCYB, while most free amino acids in HBES showed a downward trend after CotA treatment. Furthermore, CotA treatment had no influence on the types of free fatty acids and volatile flavor compounds of three Douchi samples, while the relative contents of characteristic substances (1-octen-3-ol, tetramethylpyrazine, 2,3-butanediol) generally increased after CotA treatment, indicating that CotA treatment could enrich the flavor profile of Douchi. The findings of this study demonstrated that CotA could reduce the BAs content of Douchi coupled with improve its flavor quality, providing a theoretical basis for enzyme-based control of BAs in Douchi.