Abstract: Bacillus amyloliquefaciens BI₂ isolated from straw feed was showed to be strongly antagonistic against Aspergillus flavus. Crude extract of antifungal substance was obtained by centrifugation, membrane filtration and ion exchange chromatography and it was stable at high temperature. The antifungal activity was not influenced disposing for 30min under 100℃, and only 25. 8% loss of activity was suffered. Antifungal substance, which could be steady at pH 6. 0 to pH 12. 0, was tested to have strong tolerant to several proteases, such as Trypsin, Pepsin and Papain. Finally the antifungal substance was isolated and purified by flash chromatography, and its molecular weight determined by mass spectrometry was 436. 2052u. Assay using 96-well plate illustrated that the minimum inhibitory concentration of the antifungal substance against Aspergillus flavus was 7. 8μg/mL.