Deoxynivalenol (DON), a mycotoxin produced by Fusarium species, severely contaminates grains and feed, posing a continuous threat to human and livestock health. In this study, the DON-degrading enzyme (DDE) gene from Devosia sp. JA3 was heterologously expressed in Escherichia coli. Enzyme kinetics revealed that DDE exhibited optimal activity at 37 °C and pH 7.0, with a Km of 0.32 mM and a Vmax of 563.3 nmol/(min·mg). Under optimized conditions, DDE efficiently oxidized DON to 3-keto-DON, achieving a degradation rate of 82.51% within 12 h. Further investigation in C57BL/6J mice showed that oral administration of 2 mg/kg DON significantly reduced antioxidant capacity, caused liver damage, impaired intestinal barrier function, induced intestinal inflammation and apoptosis, and disrupted the gut microbiota. DDE treatment effectively alleviated these DON-induced effects by restoring antioxidant capacity, ameliorating liver injury, downregulating pro-inflammatory and apoptotic genes, upregulating barrier-related genes, and restoring the gut microbiota balance, thereby protecting intestinal health. These findings demonstrate DDE’s excellent DON-degrading capacity and biosafety, providing new technical evidence for DON detoxification applications.