Atmospheric CO2 concentration has been proved to be increased in the future. In this study, responses of tobacco plants to stress from the tobacco aphid, Myzus persicae (Sulzer) or PVYN (potato virus Y), or stresses from both aphid and PVYN combined were investigated in OTC (open-top chambers) under ambient and elevated CO2 concentrations. We experimentally examined aboveground biomass and foliar chlorophyll, nitrogen, non-structural carbohydrates, soluble protein, total amino acid and nicotine content in tobacco plants under elevated CO2. The increase of tobacco biomass was alleviated after infected by PVYN and aphid under elevated CO2, while no significant difference of tobacco biomass was found under ambient CO2. Significant higher nitrogen content after infected with virus or by aphid + virus and soluble protein content of tobacco after infested by aphid or by aphid+virus occurred in elevated CO2. Moreover, significant higher nicotine content was observed in the tobacco with PVYN-infection in comparison with tobacco with aphids under two CO2 treatments. Our results indicated that both PVYN and tobacco aphid are major factors influencing tobacco yield and constitution significantly. The significant lower biomass of tobacco after infested by PVYN + aphid under elevated CO2 suggested that inhibition of PVYN and aphid together on tobacco growth would be stronger in future elevated CO2.Under elevated CO2 condition, both PVYN and tobacco aphid would continue to play adverse role in tobacco production. Moreover, higher CO2 concentration would strengthen the negative effects of the two integral agricultural factors.