Abstract: Objective To establish a multiplex polymerase chain reaction (PCR) assay for the rapid detection and typing of Enterotoxigenic Escherichia coli (ETEC), Enteroinvasive Escherichia coli (EIEC), Enteropathogenic Escherichia coli (EPEC), Verotoxin-producing Escherichia coli (VTEC), Eteroaggregative Escherichia coli (EAEC), Shigella and Salmonella. Methods Thirteen pairs of primers for the detection of the 7 enteropathogens with multiplex PCR were designed. After electrophoresis, the specific fragments were observed to identify the pathogens. The clinical samples were inoculated to the selective agar at 37 ℃ for 18 hours, the half clones were selected for the complex PCR. Results All the standard strains could get the positive fragments with this complex PCR assay. Among 322 clinical samples from diarrhea patients, 24 strains of Shigella, including 7 S. flexneri strains and 17 S. sonnei strains, 5 Salmonella strains, 12 EPEC strains, 6 ETEC strains, 3 EIEC strains, 1 VTEC strain and 3 EAEC strains were detected. Conclusion This complex PCR assay is rapid and specific and can detect all the forms of Escherichia coli causing diarrhea, Shigella and Salmonella in a single reaction, which could be used in the rapid detection of common diarrheal pathogens.