Objective To establish a new and rapid GeXP(GenomeLabTM eXpress Profiling)based multiplex polymerase chain reaction (PCR) assay for the detection of five common foodborne pathogens. Methods Nucleotide sequences of specific gene(invA, rfbE, PfrA, IpaH, tlh) of the five pathogens (Salmonella, Escherichia coli O157∶H7, Listeria monocytogenes, Shigella, Vibrio parahaemolyticus) were obtained and compared. The primers were then designed and the multiplex PCR assay was evaluated. Optimized assay was further validated with the detection of the unknown strains and artificially contaminated samples. Results The GeXP multiplex PCR with five sets of specific primers can be used to detect five foodborne pathogens simultaneously within 5 hours. The specificity was examined by specimens confirmed previously. The detection limit was 10³ CFU/mL. Conclusion The results suggested this GeXP multiplex PCR assay was a fast, high throughput test for foodborne bacterial pathogens.