Abstract: Aflatoxin B₁ (AFB₁) is a highly toxic and carcinogenic mycotoxin that poses a serious threat to food safety and human health. This study aimed to investigate the degradation characteristics of Aflatoxin B₁ (AFB₁) by the laboratory-preserved strain Bacillus aerius CGMCC No.31025 and to identify its degradation products. Through various treatments of fermentation broth, studies on active components, and physicochemical properties of active substances, the primary active components of B. aerius CGMCC No.31025 degrading AFB₁ were determined using high-performance liquid chromatography (HPLC). The degradation products were separated and identified via ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Results indicated that the primary active component responsible for AFB₁ degradation by B. aerius CGMCC No.31025 was the fermentation supernatant, which exhibited high thermal stability for its effective active substances. Protein inactivation treatments indicated that the active component degrading the toxin in the fermentation supernatant may be an enzyme or other active protein. UHPLC-MS/MS analysis identified nine degradation products, which were predicted to result primarily from reduction and dealkylation of functional groups in the parent AFB₁ structure. This study provides potential microbial strains and technical support for the biological removal of AFB₁ contamination in food and feed.