In order to explore the application prospects of DNA barcode technology in microbial identification, the 16S rRNA sequences of 8 common pathogenic microorganisms in food were compared. The PCR amplification, sequencing and database search were conducted followed by universal primers designing, which were purposed to establish a microbial identification method based on DNA barcode. The results showed that the variable region sequence of V3～V6 amplified provided sufficient resolution for Gammaproteobacteria Enterobacteriales, including Vibrionales, and Pseudomonadales to accurately distinguish pathogenic bacteria and related interfering bacteria, corresponding to Bacillibacteria (Bacillales) could be identified to the “genus” level.