Ebola is an emerging infectious disease caused by a deadly virus belonging to the family Filoviridae， genus Ebolavirus. Based on their geographical distribution， Ebolavirus has been classified into total five species so far， mainly Zaire， Sudan， Taï Forest， Bundibugyo and Reston. It is important to be able to differentiate the Ebolavirus species as they significantly differ in pathogenicity and more than one species can be present in an area. We have developed a one-step step-down RT-PCR detecting all five Ebolavirus species with high sensitivity (1 copy of Ebolavirus DNA， 10 copies of RNA and 320 copies of RNA spiked in 1 ml whole blood). The primers and FRET-probes we designed enabled us to differentiate five Ebolavirus species by distinct Tm (Zaire: flat peaks between 53.0°C and 56.9°C; Sudan: 51.6°C; Reston: flat peaks between 47.5°C and 54.9°C; Tai Forest: 52.8°C; Bundibugyo: dual peaks at 48.9°C and 53.5°C)， and by different amplicon sizes (Zaire 255 bp， Sudan 211 bp， Reston 192 bp， Taï Forest 166 bp， Bundibugyo 146 bp). This one-size-fit-all assay enables the rapid detection and discrimination of the five Ebolavirus species in a single reaction.