This review describes the development of the multiplex PCR detection kit for Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in food samples. To develop a detection assay, our research team evaluated the optimization of the pre-enrichment broth, the simple DNA extraction method, and the multiplex PCR settings. When this detection protocol was used to detect the above pathogenic bacteria, one cell per 25 g of inoculated sample was detected within 24 h. Moreover, there was excellent agreement between the multiplex PCR assay and the conventional culture method. The multiplex PCR detection assay system was confirmed to be a reliable and useful method for the rapid screening of food products for foodborne pathogens. The assay system was commercialized as a “[TA10] Pathogenic Bacterial Multiplex PCR Detection Kit”. When this kit was provided to four different laboratories for an extensive validation study, there were no significant differences in detection sensitivity among the laboratories. The detection kit will be valuable as a screening method for foods contaminated with these pathogens, and it will also be useful for identifying the sources of outbreaks of foodborne illness.