Enteric pathogenic E. coli have been broadly classified into six pathovars due to the specific colonization factors and virulence factors; enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), diffusely adherent E. coli (DAEC), and enterohaemorrhagic E. coli (EHEC). While diagnostic methods mainly focused on the target genes of EHEC, it was reported that ETEC caused the food borne disease 2012 outbreak in Korea. The diagnostic method was required to be able to rapidly detect the other pathogenic E. coli as well as EHEC. To develop a rapid and sensitive detection method, a microfluidic chip-based real-time PCR approach has been developed. Twenty one target genes of pathogenic E. coli such as EPEC(bfpU, eae), ETEC(eltA, eltB, sta), EIEC(icsA, gtrA, gtrB, gtrII), EHEC(stx1A, stx1B, stx2A, stx2B, hlyA, hlyB, hlyC, hlyD), EAEC(aggR, astA) and DAEC(afaE-1, afaE-3) were selected to design target specific primers. By using these target genes, we were able to detect pathogenic E. coli within 30 minutes. This method has an ability to detect virulence factors rapidly and widely for samples related to each type of E. coli infection. Therefore, the method developed here may be used for the upcoming food borne illness investigation.