Abstract: Aflatoxin B₁ (AFB₁) and zearalenone (ZEN) are prevalent fungal contaminants in grains and feed, posing significant risks to human and animal health. In recent years, enzyme-based biodegradation has emerged as a safe and efficient strategy for mitigating mycotoxin contamination. This study utilized BlastP analysis to identify candidate mycotoxin-degrading enzymes, which were subsequently characterized for their enzymatic properties. Molecular docking was performed to analyze interactions between the enzyme and its substrates, while UHPLC-MS/MS was employed to identify degradation products. Additionally, the toxicity of these degradation products was assessed using a zebrafish liver toxicity model. The findings revealed that a novel CotA laccase, designated as BH CotA, was successfully identified from Bacillus haynesii, with a molecular mass of 59.4 kDa. BH CotA demonstrated robust degradation activity toward both AFB₁ and ZEN under conditions of pH 8.0 and 70 ℃. The enzyme retained over 90% of its initial activity after 1 h of incubation across a temperature range of 30-70 ℃ and a pH range of 8.0-10.0, highlighting its excellent thermal and pH stability. UHPLC-MS/MS analysis confirmed that BH CotA oxidized AFB₁ into AFQ₁ and converted ZEN into 15-OH-ZEN. Zebrafish liver toxicity assays further demonstrated that both AFQ₁ and 15-OH-ZEN exhibited significantly reduced toxicity compared with their parent compounds. Collectively, this study introduces a promising enzymatic bioremediation approach for the effective detoxification of AFB₁ and ZEN in food and feed systems, laying a foundation for the development of safe and practical mycotoxin control strategies.