An effective, multiplex real-time PCR assay for the simultaneous detection of three major pathogens, Nosema bombycis (BmNb), nucleopolyhedrovirus (NPV) and densovirus (DNV), in B.mori was developed in this study.BLAST searches revealed that the primers and probes used in this study had high specificities for their target species.The ability of each primerprobe set to detect pure pathogen DNA was determined using a plasmid dilution panel, in which under optimal conditions the multiplex real-time PCR assay showed high efficiency in the detection of three mixed target plasmids with a detection limit of 2.5× 10-5 ng for BmNb, B.mori NPV (BmNPV) and B.mori DNV (BmDNV).When the ability to detect these three pathogens was examined in artificially inoculated silkworms, our method presented a number of advantages over traditional microscopy, including specificity, sensitivity and high-throughput capabilities.The multiplex real-time PCR assay showed early detection of BmNPV and BmDNV by day 1 post inoculation (p.i.) using DNA templates of the three pathogens in various combinations from individually infected silkworms;the early detection of BmNb was possible by day 3 p.i.using the DNA isolated from the midgut ofN.bombycis-infected silkworms.