Bacillus subtilis ZJ-2019-1 effectively detoxifies aflatoxin B1 (AFB1) and zearalenone (ZEN) simultaneously. The strain was isolated from pig feces and demonstrated high degradation efficiency under optimal conditions. At 37°C and pH 7.0, B. subtilis ZJ-2019-1 reduced AFB1 by 60.88% and ZEN by 33.18% within 72 hours. Autoclaved and boiled supernatants enhanced degradation, achieving 79.85% and 100% for AFB1 and ZEN, respectively, at 1 mg/L within 48 hours. Crude enzymes from the strain degraded AFB1 and ZEN by 100% and 94.29% within 72 hours at 70°C. Divalent cations like Co²⁺, Fe²⁺, Mn²⁺, and Ni²⁺ significantly enhanced degradation activity. When applied to corn gluten meal, B. subtilis ZJ-2019-1 detoxified 66.08% of AFB1 and 22.01% of ZEN, surpassing a commercial detoxification agent. The study highlights the potential of B. subtilis ZJ-2019-1 as a promising candidate for simultaneous removal of multiple mycotoxins in food and feed, addressing health concerns associated with harmful mycotoxins. The strain's active components are primarily found in its supernatants, which show enhanced degradation under alkaline conditions and in the presence of certain metal ions. The findings suggest that B. subtilis ZJ-2019-1 is a valuable tool for mycotoxin detoxification in agricultural and food applications.Bacillus subtilis ZJ-2019-1 effectively detoxifies aflatoxin B1 (AFB1) and zearalenone (ZEN) simultaneously. The strain was isolated from pig feces and demonstrated high degradation efficiency under optimal conditions. At 37°C and pH 7.0, B. subtilis ZJ-2019-1 reduced AFB1 by 60.88% and ZEN by 33.18% within 72 hours. Autoclaved and boiled supernatants enhanced degradation, achieving 79.85% and 100% for AFB1 and ZEN, respectively, at 1 mg/L within 48 hours. Crude enzymes from the strain degraded AFB1 and ZEN by 100% and 94.29% within 72 hours at 70°C. Divalent cations like Co²⁺, Fe²⁺, Mn²⁺, and Ni²⁺ significantly enhanced degradation activity. When applied to corn gluten meal, B. subtilis ZJ-2019-1 detoxified 66.08% of AFB1 and 22.01% of ZEN, surpassing a commercial detoxification agent. The study highlights the potential of B. subtilis ZJ-2019-1 as a promising candidate for simultaneous removal of multiple mycotoxins in food and feed, addressing health concerns associated with harmful mycotoxins. The strain's active components are primarily found in its supernatants, which show enhanced degradation under alkaline conditions and in the presence of certain metal ions. The findings suggest that B. subtilis ZJ-2019-1 is a valuable tool for mycotoxin detoxification in agricultural and food applications.