Virus-induced gene silencing (VIGS) showed several advantages to identify gene functions such as short experimental cycle, more broad hosts, etc. In this study, the feasibility and efficiency of employing Barley stripe mosaic virus (BSMV)-based VIGS system to evaluate Fusarium head blight (FHB) resistance were explored in wheat. With variable conditions tested, it showed that the maximal silencing efficiency 78% on spike was obtained when the recombinant BSMV was inoculated on flag leaf at flagging stage. However, the plant may reduce its own immunity to FHB when inoculated with BSMV. To induce this impact, different Fusarium graminearum strains were tested and SF06-1 strain was selected for FHB resistance evaluation. Using this system, TaAOC, TaAOS, and TaOPR3 involved in jasmonic acid (JA) signaling pathway were identified to positively regulate FHB resistance, which was underpinned by the results when silencing TaAOS in wheat by stable transgenic plants.