Abstract: Objective To develop a duplex fluorescence RT-PCR assay for detecting four pathogenic subtypes of Ebola virus, including the subtypes of Sudan, Zaire, Bendi bujiao and Ivory Coast.Methods Based on the conserved sequence of nucleoprotein (NP) gene of four subtypes of Ebola virus, two sets of primers and probes were designed corresponding to the Sudan/Zaire subtype and to Bendi bujiao/Ivory Coast subtype. Using the Ebola virus RNA of four subtypes transcribed in vitro as templates, the reaction condition were optimized and the specificity, sensitivity, and reproducibility were tested. Then a duplex fluorescence RT-PCR assay was developed. Results The specificity of the method was high on detection the Ebola virus nucleic acid. There were no cross reactions with hemorrhagic fever with renal syndrome virus, Dengue virus, yellow fever virus, West Nile virus, Japanese encephalitis virus and Chikungunya virus. Detection sensitivities of two sets of primers and probes can reach a minimum of 50 copies/μL. Duplicate detections for three times had better repeatability at four dilutions (2×108, 2×106, 2×104, 2×102 copies/μL) of positive control RNA templates of subtypes of Sudan and Bendi bujiao. Conclusion The duplex fluorescence RT-PCR assay can simultaneously detect the four subtypes of Ebola virus with high sensitivity and specificity, and can be used for detection on suspected cases of Ebola virus infection.