To identify the 16S-23S rRNA gene in Enterobacter sakazakiis, a PCR-DHPLC assay was performed in this study. Primers specific for the 16S-23S rRNA gene of Enterobacter sakazakii in food were selected to conduct the DHPLC assays. The specific testing was performed with Enterobacter sakazakii and 59 strains, and various grads of Enterobacter sakazakii was used for the sensitivity testing. The good specificity was found in the study. Also, the method showed nicer sensitivity with the lowest amount of detecting was 181 CFU/mL, so it can detect and identify Enterobacter sakazakii quickly and correctly. The DHPLC could be a new method for detecting food pathogens.