Abstract: In order to efficiently identify the main viruses, including Tobacco mosaic virus(TMV), Cucumber mosaic virus(CMV), Potato virus Y(PVY)and Tobacco vein banding mosaic virus(TVBMV), of tobacco, a multiplex RT-PCR approach for simultaneous detection of four viruses was established. Through inoculating the four viruses, the antagonism of TMV, CMV and PVY on TVBMV in mixed infection was discovered. In order to improve the detection sensitivity for TVBMV in mixed infection, PIPO with the highest expression level in TVBMV was screened out. By using the primers designed on the basis of the conservative sequences of PIPO gene of TVBMV and CP genes of TMV, CMV and PVY and optimizing the concentration of primers and templates, the fragments with sizes of 700, 452, 275 and 235 bp were amplified stably in one PCR reaction system. This method enables the simultaneous detection of the four viruses and is suitable for monitoring multiple virus infection in most tobacco growing areas.