In order to rapidly screen for the virulence factor that produces pathogenic Escherichia coli in food, we have developed multiplex polymerase chain reaction (PCR) assays. The multiplex PCR assays detect 4 pathogenic genes of enterohaemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), and enteroinvasive E. coli (EIEC). This allows for the generation of specific fragments 150, 179, 218, 401, 465, 584, and 881 bp for VT1, ST, LT, 16S rRNA, inV, VT2, and eaeA genes, respectively. The detection limit of 3 log CFU/mL for eaeA, LT, VT1, VT2, 4 log CFU/mL for inV, 6 log CFU/mL for ST by single PCR, while 5 log CFU/mL for VT1, VT2, 6 log CFU/mL for eaeA, LT, 7 log CFU/mL for ST, inV by multiplex PCR. This optimized detection method of pathogenic E. coli can be used as supportive data to revise the microbiological analytical manuals for the Korean Food Code.