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Aflatoxins, widely found in feed and foodstuffs, are potentially harmful to human and animal health because of their high toxicity. In this study, a strain of <i>Bacillus amyloliquefaciens</i> B10 with a strong ability to degrade aflatoxin B1 (AFB1) was screened; it could degrade 2.5 μg/mL of AFB1 within 96 h. The active substances of <i>Bacillus amyloliquefaciens</i> B10 for the degradation of AFB1 mainly existed in the culture supernatant. A new laccase with AFB1-degrading activity was separated by ammonium sulfate precipitation, diethylaminoethyl (DEAE) and gel filtration chromatography. The results of molecular docking showed that B10 laccase and aflatoxin had a high docking score. The coding sequence of the laccase was successfully amplified from cDNA by PCR and cloned into <i>E. coli</i>. The purified laccase could degrade 79.3% of AFB1 within 36 h. The optimum temperature for AFB1 degradation was 40 °C, and the optimum pH was 6.0–8.0. Notably, Mg²⁺ and dimethyl sulfoxide (DMSO) could enhance the AFB1-degrading activity of B10 laccase. Mutation of the three key metal combined sites of B10 laccase resulted in the loss of AFB1-degrading activity, indicating that these three metal combined sites of B10 laccase play an essential role in the catalytic degradation of AFB1.