The invention belongs to the field of the environmental biotechnology, and specifically relates to an aspergillus flavus strain incapable of producing aflatoxin and an application of the aspergillus flavus strain in biological prevention and control of aflatoxin pollution of peanuts. The aspergillus flavus strain incapable of producing aflatoxin is named as aspergillus flavus NAFFHN396, and is preserved in the China General Microbiological Culture Collection Center (CGMCC) on May 28, 2015, and the preservation serial number is CGMCC No: 10927. The aspergillus flavus strain incapable of producing aflatoxin with deletion of an aflatoxin synthetic cluster gene is obtained, and has an obvious inhibiting effect for the yield of the toxin of the strain with strong capacity of producing aflatoxin, thus a biocontrol bacterium source is provided for the biological prevention and control of the toxic aspergillus flavus generated in the field in the future, the aspergillus flavus strain is separated from China, has good colonization capacity in peanuts and potential field competitive advantage, and has great significances in inhibiting infection of the peanuts by the toxin production aspergillus flavus, and reducing the pollution of the aflatoxin in the peanuts.