Abstract: Objective To develop a multiple PCR-based reverse line blot hydrization assay （mPCR-RLB） to simutaneously detect several STD pathogens: Neisserria gonorrhoeae, Chlamydia trachomatis, Ureaplasma urealyticum, U. parvum, Mycoplasma genitalium, M. hominis and Trichomonas vaginalis. Methods Seven pairs of biotin-labelled primer were designed and synthesized to target the 16S rRNA-23S rRNA intergenic spacer regions of Neisserria gonorrhoeae, Chlamydia trachomatis, Mycoplasma, and repetitive DNA sequence of Trichomonas to identify and subtype these pathogens. DNA was extracted from the referrence strains of seven pathogens and used as templates. mPCR was performed to simutaneously amplify the target regions of these pathogens. Then, the biotin-labelled amplicons were hybridized with membrane- bound specific oligonucleotide probes followed by the detection of bound amplicons with chemiluminescence assay. Serially diluted plasmids containing the target genes of pathogens were amplified with this method to detect its sensitivity. Two-hundred and eleven specimens, including 104 male urethral swabs and 107 female cervical swabs, were collected from the STD clinic of Wuhan First Hospital; mPCR-RLB and single-primer PCR were performed. For specimens with inconsistent results, nested PCR was performed to confirm the results. Results The assay sensitively and specifically identified referrence strains of the tested pathogens. The detection limit of mPCR-RLB was 100 copies for all the pathogens. Of the 211 clinical specimens, 2.8% （6/21） were negative for single-primer PCR, but positive for mPCR-RLB, and nested-PCR results were consistent with those of mPCR-RLB. Conclusion mPCR-RLB is a sensitive, specific and rapid method for the detection of STD pathogens from clinical specimens.