The specificity of this method was evaluated by testing 17 species closely related bacteria in the laboratory, and the amplification product was analyzed by the Tm value of the dissolution curve, which had good specificity and could detect five different virulence genes: aggR for EAEC, eae for EHEC and EPEC, LT for ETEC, ipaH for EIEC in the same reaction tube. The fecal simulated samples showed that the sensitivity reached 10⁴~10⁶ CFU/mL. The multiplex real-time PCR detection method established in this experiment is suitable for the preliminary screening of five kinds of Escherichia coli.