Abstract: Objective To establish a multiplex RT-PCR assay for the simultaneous detection of Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Bauman acinetobacter, Coagulase negative staphylococcus, Staphylococcus aureus and Candida albicans in the blood of sepsis patients. Methods Primers and probes were designed according to the conserved sequence of the pathogenic bacteria or fungi to establish a multiplex RT-PCR assay. To analyze the sensitivity and specificity of the multiplex RT-PCR assay established, 112 sepsis positive blood specimens (blood culture and biochemical identification) were tested by using the assay. Results A multiplex RT-PCR assay of common pathogenic bacteria and fungi causing sepsis was established successfully. This method had good specificity and sensitivity, the detection sensitivity was 10-5 ng/l. Compared with the standard detection method (blood culture plus biochemical identification),20 hours were reduced. Among 83 target pathogens, 80 were detected to be positive by multiplex RT-PCR. Among 29 non target pathogens, no positive results were obtained. The specificity and sensitivity were 100% and 96.39% respectively. Conclusion The multiplex RT-PCR assay is rapid, efficient, sensitive and specific, which could be applied in the detection of common pathogens in the blood of ICU patients.