To develop a multiplex PCR protocol by which Escherichia coli and Salmonella in raw pork could be simultaneously detected and investigate possible pathogenic bacteria contamination.Methods Primers were designed in accordance with the rfbE gene of Escherichia coli O157∶H7 and invA gene of Salmonella. Through reaction optimization, specificity and sensitivity tests of single gene and multiplex gene PCR, a duplex PCR method was established for rapid detection of Escherichia coli and Salmonella. 144 raw pork samples were collected from different general supermarkets, chilled meat shops and farmer's markets in the city of Zhengzhou, and were analyzed by the PCR method. Conventional microbiological examination was also taken as a control. Results The duplex PCR protocol turned out to be specific, effective with a sensitivity of 10 pg/μl. The examination of the raw pork samples showed that 10 out of 144 were Escherichia coli positive (6.94%), whilst Salmonella was detected among 13 samples (9.03%), and 2 samples were both positive (1.38%).Conclusion A duplex PCR assay was established for the rapid simultaneous detection of Escherichia coli and Salmonella, and pathogenic bacteria contamination existed in raw pork which should be concerned about.