Abstract: In order to prevent the mildewing of traditional beef jerky in the early stage of retention period, a PCR assay for detection of molds in beef jerky was established, and the specificity and sensibility of the PCR assay were verified. The mouldy fresh beef, yeast, Lactococcus lactis, Lactobacillus and E. coli were used as materials to verify the specificity of the established PCR assay, and a series of different concentration gradients of DNA solutions extracted from mouldy fresh beef were used to identify its sensibility. The results demonstrated that when the DNA extracted from yeast was used as a template, a specific 300 bp amplicon was obtained by the PCR assay; however, no amplicons were obtained using the DNA extracted from other samples as template, which indicated the good specificity of the established PCR assay. The specific bands were observed when the DNA stock solution extracted from mouldy fresh beef samples as well as the 10 times, 100 times and 1 000 times diluent of the stock solution were used as template in the PCR assay. The 2 ng/μL of the DNA template was still sensitive to the primers. The PCR assay developed in this study provided a new technique for the rapid detection of molds contamination of saled traditional beef jerky in market.