Tomato is cultivated worldwide as a nutrient-rich vegetable crop. Tomato wilt disease caused byFusarium oxysporumf.sp.Lycopersici(Fol) is one of the most serious fungal diseases posing threats to tomato production. Recently, the development of Spray-Induced Gene Silencing (SIGS) directs a novel plant disease management by generating an efficient and environmental friendly biocontrol agent. Here, we characterized thatFolRDR1(RNA-dependent RNA polymerase 1) mediated the pathogen invasion to the host plant tomato, and played as an essential regulator in pathogen development and pathogenicity. Our fluorescence tracing data further presented that effective uptakes ofFolRDR1-dsRNAs were observed in bothFoland tomato tissues. Subsequently, exogenous application ofFolRDR1-dsRNAs on pre-Fol-infected tomato leaves resulted in significant alleviation of tomato wilt disease symptoms. Particularly,FolRDR1-RNAi was highly specific without sequence off-target in related plants. Our results of pathogen gene-targeting RNAi have provided a new strategy for tomato wilt disease management by developing an environmentally-friendly biocontrol agent.