The invention discloses a technology of producing an aflatoxin B1 (AFB1) degradation preparation by mixed fermentation and application. Microorganisms used by the AFB1 degradation preparation preparedby the invention comprise aspergillus oryzae CGMCC 3.800, extracellular enzyme secreted by saccharomyces cerevisiae ATCC 204508, an amboceptor, an adjuvant and the like. The extracellular enzyme of the preparation comprises working enzyme (glucose oxidase, polyphenol oxidase) capable of degrading AFB1, assisting enzyme (pectinase, xylanase, cellulase) for improving degradation efficiency, and also comprises micromolecule amboceptor for transferring electron, adjuvant and the like. The working enzyme is oxidase, a catalytic process involves electron transfer and free radical generation, the micromolecule amboceptor and adjuvant can carry the electron into the interior of tissue of grain and oil, feed and related products, so as to degrade the AFB1 infected into the interior of tissue, namely multi-path and multi-step catalysis, thus realizing thorough detoxification of the AFB1. The preparation is convenient to use, small in dosage, high in biological security coefficient, and has gooddegradating effect on the AFB1 in mildewed grain and oil, feed and related products.