Primers, and probes were designed for the conserved sequences of five virulence genes involved in Escherichia coli diarrhea mentioned in the new standard of GB 4789.6—2016 national food safety standard. Four kits of multiple real-time fluorescence PCR were established to detect five diarrhea-causing E. coli simultaneously. The results showed that 12 pairs of virulence genes of the four kits were verified by the standard reserve strains of the five diarrhea-causing E. coli strains. In addition, these 12 pairs of virulence genes only play a role in the specific role of corresponding diarrhea-causing E. coli, and have no amplification curve for common foodborne pathogens. The detection limit of escV, bfpB, stx₁, ipaH and invE genes is 10 CFU/mL, and that of aggR gene is 10² CFU/mL. The detection limit of astA and Pic genes was 10³ CFU /mL. When the concentration of stx₂A, ipaH and stIb genes was less than 10 CFU/mL, a significant “S” type amplification curve could be observed, and the line shape was better. In addition, 40 samples including meat, milk, animal diarrhea, and some artificial contamination samples were tested, and a total of 11 positive samples were found. Consistent with the test results of the new standard 4789.6—2016, The results showed that the four kit methods were simple, specific, sensitive and practical.