Salmonella enterica serovar Heidelberg,（SH）is pathogenic serotypes within Salmonella. Using BLAST and PCR validation for all CDSs of SH, four serovar-specific genes were identified including SeHA_C2639, SeHA_C2640, SeHA_C3259 and SeHA_C3258. A PCR protocol was developed by pHAm8 (350 bp) of SH-specific primer and 139-141 (284 bp) of Salmonella-specific primer and evaluated for the detection of SH. The result showed that the 55 strains of serotype of Salmonella and other common foodborne pathogens proved that the PCR assay had very good specificity for the detection of SH. The detection limit was 6.1 × 10² CFU/mL in pure culture. It was shown that S. Typhimurium and Escherichia coli as interfering bacteria up to concentrations of N×105~N×101 CFU/mL, respectively, did not interfere with PCR detection of SH. In artificially contaminated milk, the protocol could detect less than 1.52 CFU/mL after 8 h enrichment. The detection method could quickly and accurately detect food SH, application in the field of food safety in favor.