Aflatoxin B (AFB ) removing bacterial strains were isolated from different habitats that were easily contaminated by AFB . Furthermore, the composition of the fermentation medium and conditions of fermentation process were optimized, including carbon source, nitrogen source, metal ions, temperature, initial pH value, inoculation volume, culture broth volume. Using coumarin as the sole carbon and energy source, we primarily screened 31 strains, and 10 strains were found to be capable to remove AFB . Among them, where the highest removal rate of 71.91% appeared in those isolated from rotten wood (poplar). Strains XY1, XY3 and T6 were carried out to identify, and the results were Klebsiella sp., Klebsiella pneumonia, and Pantoea sp. respectively. Corn cob powder and tryptone can significantly increase the AFB removal activity of these strains. The AFB removal activity of Klebsiella sp.XY1 and Klebsiella pneumonia XY3 can be enhanced by Ca , and those of Pantoea sp.T6 can be enhanced by Cu . Temperature and initial pH were positively correlated with the AFB removal activity of these strains in a certain range. This study not only provides reference for the screening and application of AFB removing bacteria, but also provides a basis for possible application in the food and feed industry.