Abstract: In order to attain the aflatoxin B₁ (AFB₁) degrading strains, in this study, strains with the capacity of degrading AFB₁ was obtained from straw, cow dung, soil, moldy corn and peanuts using coumarin as the sole carbon source in the selected medium. The physiological and biochemical analysis combined with 16S rDNA gene sequence analysis were used to verify the species of the strains; the optimal components of the fermentation medium was investigated; the contents with the ability to degrade AFB₁ was determined using protease K and SDS treatment; the protein profiles were illustrated using SDS-PAGE analysis and the different protein bands were analyzed using LC-MS; the degrading compounds were extracted and full wavelength scanning and liquid-mass spectrometry analysis were employed for further analysis; the possible formulas of the degraded compounds were inferred based on its molecular mass. The results indicated that the strain Klebsiella sp N1 in DJN medium showed the best effect on aflatoxin degradation, with a degradation rate of 74% within 72 hours at 37℃ detected by high performance liquid chromatography (HPLC). Meanwhile, the aflatoxin degradation rates of the fermentation supernatant of SC and DPN medium were 58% and 36% respectively and the aflatoxin absorbance of bacteria biomass was 20%. After treatment of fermentation supernatant with protease K and SDS, the degradation rate of AFB₁ was significantly reduced, which were 10.5% and 0%, respectively. These phenomena suggested that AFB₁ might be degraded by extracellular enzymes. SDS-PAGE electrophoresis illustrated that the protein content in the supernatant of DJN was higher than that in SC and DPN medium. The different protein bands analysis indicated that the protein might be Mn-superoxide dismutase (SodA) or NADH-quinone redox enzyme (NuoB) with molecular mass 23 kDa. The degradation products were extracted using chloroform, ethyl acetate and n-butanol, the full wavelength scanning results showed that the products could be leach by n-butanol. In addition, the full wavelength scanning of the degradation products showed that the maximum absorption peak was at 310 nm, and its molecular weight was 241.1 after extraction by n-butanol and analyzed by LC-MS/MS. The results might suggest that 8-and 9-digit double bonds on the furan ring and the 12-digit methoxy group might be destroyed in aflatoxin B₁ according to the molecular mass of the degraded compounds.