Abstract: One-step multiplex RT-PCR combines reverse transcription(RT) with polymerase chain reaction(PCR) in to one step which amplifies a number of targets simultaneously in one reaction system.For the purpose of simultaneously detecting three viruses(Cucumber mosaic virus,Tobacco mosaic virus and Potato virus Y) in tobacco with the said method,three pairs of specific primers were designed according to the relatively conservative regions of the three viruses,and the reaction system and conditions of RT-PCR were optimized. The results of optimization showed that the final multiplex RT-PCR featured higher reaction specificity and the amplification efficiencies of the three fragments were basically the same.The detection results of samples from field experiments indicated that one-step multiplex RT-PCR could fast and accurately detect the three viruses simultaneously,it was more competent than ELISA(Enzyme-linked immunosorbent assay).