Abstract: To investigate the active substances of aflatoxin B₁ (AFB₁) degradation in Bacillus sonorensis s262, the fermentation conditions for AFB₁ degradation by strain s262 were optimized based on a single-factor test combined with response surface methodology. The proteins in the fermentation supernatant were precipitated using ammonium sulfate, and the crude enzyme solution obtained was subjected to mass spectrometry analysis and enzyme activity assay. The results showed that: the degradation active substances were mainly derived from the extracellular metabolites of the strains. The optimal fermentation conditions were 6% inoculum of seed solution, 48 h fermentation time, and 38 ℃ fermentation temperature. The catalase activity derived from the mass spectrometry assay showed a significant positive correlation with the AFB₁ degradation rate (P<0.01). Therefore, it is hypothesized that catalase is the main active substance in the degradation of AFB₁ by the fermentation broth of strain s262.