A universal protocol for PCR detection of 13 species of foodborne pathogens in foods was developed. The protocol used a universal enrichment medium and same PCR conditions with 13 sets of specific primers. The 13 species of foodborne pathogens examined were Escherichia coli (all), E. coli-ETEC, E. coli-O157:H7, Shigella, Salmonella, Yersinia enterocolitica, Y. pseudotuberculosis, Vibrio cholerae, V. parahaemolyticus, V. vulnificus, Listeria monocytogenes, Staphylococcus aureus and Bacillus cereus. No interference was observed using the PCR assay when food samples were artificially inoculated with each individual bacterial species. Twelve different seafood samples and 2 soft cheese samples without artificially inoculation were examined by this protocol. Vibrio vulnificus, Salmonella, E. coli, Listeria monocytogenes, and Bacillus cereus were found to be contaminated in some foods.