Abstract： Objective Marburg virus and Ebola virus are acute infections with high case fatality rates.A rapid,sensitive detection method was established to detect Marburg virus and Ebola virus by multiplex real-time fluorescence quantitative PCR.Methods Designing primers and Taqman probes from highly conserved sequences of Marburg virus and Ebola virus through whole genome sequences alignment,Taqman probes labeled by FAM and Texas Red,the sensitivity of the multiplex real-time quantitative PCR assay was optimized by evaluating the different concentrations of primers and Probes.Results We have developed a real-time PCR method with the sensitivity of 30.5 copies/μl for Marburg virus positive plasmid and 28.6 copies/μl for Ebola virus positive plasmids,Japanese encephalitis virus,Yellow fever virus,Dengue virus were using to examine the specificity.Conclusions The Multiplex real-time PCR assays provide a sensitive,reliable and efficient method to detect Marburg virus and Ebola virus simultaneously.