Abstract: Cucumber mosaic virus (CMV) disease and potato virus X (PVX) disease major diasesese that cause great loss in tobacco production. In order to prevent CMV and PVX an attenuated vaccine was constructed which could prevent CMV and PVX simultaneously. A mutant of CMV RNA2 named pCB301-CMV_Fny-R2-2bPT was constructed firstly in which the expression of 2b protein was terminated prematurely. Then an 200 bp conserved sequence (5890-6089) of PVX was inserted into pCB301-CMV_Fny- R2-2bPT and obtained the mutant named pCB301-CMV_Fny-R2-2bPT-PVX_5890-6089. The pCB301-CMV_Fny-R2-2bPT-PVX_5890-6089 plasmid mixed with the plasmid containing the wild type CMV_Fny-RNA1 and CMV_Fny-RNA3 in the same amount was used to inoculate Zhongyan 100, Hongda and Nicotiana sylvestris with injection of Agrobacterium. The inoculated plants showed weak infectivity compared with the symptoms of wild type CMV_Fny-RNA1+RNA2+RNA3 mixed inoculation. The stability test of the vaccine confirmed that the inserted PVX 200 bp conservative fragment could exist stably. The experiments of CMV and PVX intensity strains showed that the attenuated vaccine could cross protect CMV and PVX of Zhongyan 100 simultaneously.