To develop a method for identification of B-type fumonisin-producing Aspergillus nigerat genetic level. Methods The DNA and mRNA extraction method, conditions fo ramplification of eight key genes of B-type fumonisin-producing gene cluster of Aspergillus niger by polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR) were optimized. The expression products were analyzed. Results The DNA of eight key genes related to B-type fumonisin biosynthesis were detected in all 19strains of Aspergillus niger. fum6,fum14,fum19 were the most predominant genes in Aspergillus niger in association with fumonisin production, but the mRNA expression level of the 8key genes were different which leaded to different B-type fumonisin produce ability.Conclusion B-type and non-B-type fumonisin-producing Aspergillus nigercould not be distinguished at DNA level. DNA in combination with mRNA expression level was needed to identify B-type fumonisin-producing Aspergillus niger.