【Aim】The fusion TEER-decreasing protein (fTDP) induces tobacco resistance to tobacco mosaic virus (TMV). To enhance the field efficacy and stability of fTDP, we optimized the combination ratios of agents and explored the induction mechanism. 【Method】Using Nicotiana tabacum var. Samsun NN and N. tabacum K326 as materials, we analyzed the number of necrotic spots, disease symptoms, and expression levels of the TMV coat protein gene (TMV-CP) in tobacco plants treated with different agents to identify the most effective antiviral agent. The optimal ratio of the selected agent, ningnanmycin, to fTDP was determined via the co-toxicity coefficient method. The disease resistance efficacy and resistance pathways of the fTDP/ningnanmycin combined agent were evaluated based on indicators such as plant growth status, defense enzymes, and defense genes. The field application efficacy of the combined agent was measured with Yunyan 87 as the material. 【Result】Ningnanmycin and fTDP delayed the appearance of mosaic symptoms of tobacco and significantly reduced the viral load. When the mass ratio of fTDP to ningnanmycin was 3 : 7, the combined agent exhibited a significant synergistic effect of TMV inactivation, with a co-toxicity coefficient of 152. When the mass ratio of fTDP to ningnanmycin was 1 : 9, the combined agent protected tobacco K326 from TMV infection, with a significant synergistic effect. In addition, the combined agent increased salicylic acid content, defense enzyme activity, and expression levels of multiple disease resistance-related protein genes in tobacco K326. Field trial results demonstrated that the combined agent at this ratio exhibited higher and more stable efficacy than the single agents, achieving the control efficacy of 77.8%. 【Conclusion】The fTDP/ningnanmycin combined agent enhances tobacco resistance to TMV by activating the salicylic acid signaling pathway. When the mass ratio of fTDP to ningnanmycin was 1 : 9, the combined agent outperformed single agents in both antiviral activity and cost reduction in both laboratory and field settings.