Abstract: Aspergillus flavus is a ubiquitous fungal pathogen in the worldwide.Early and accurate detection of A.flavus is essential to reduce the damage of Aflatoxins produced by A.flavus.Based on the difference of rDNA ITS sequences among A.flavus and other Aspergillus spp., apair of specific primers, S1/X2, was designed in this study.The primer amplified a single 334bp product from all isolates of A.flavus and that were not from other four Aspergillus species and 17 other fungi and bacteria isolates tested.The detection sensitivity was 280fg of genomic DNA.The PCR-based detection method developed here could also be used to detect A.flavus from naturally infected peanut or corn tissues.