A degrading enzyme PADE produced during the degradation of aflatoxin B1 (AFB1) by the laboratory screening bacteria M19 was selected as the subject in this study. Thin layer chromatography and fluorescence spectrum analysis were performed to analyze possible structural changes of products in the AFB1 degradation solution. Mass spectrometric were further used to detect the AFB1 degradation products. The results of thin layer chromatography showed that no new fluorescent absorbent substance was detected in the organic phase degradation solution, and no absorbent substance was detected in the aqueous phase degradation solution, indicating that the degradation product did not have fluorescence absorption. The fluorescence spectrum detection results showed that the fluorescence of the AFB1 degradation product was decreased obviously. The mass spectrometry results showed that a substance P with a mass-to-charge ratio of 227.18 was found. Based on all the results, it can be concluded that the lactone bond was broken during the degradation of AFB1, and a degradation product P with a molecular weight of 226 was generated. The molecular formula was C14H10O3 as analyzed by Xcalibur software. The degradation pathway was hypothesized.