Tilapia lake virus (TiLV) is highly contagious and pathogenic to tilapia, causing enormous economic losses in tilapia industry. Up to now, there is no commercial vaccine is available for against TiLV infection. Therefore, a novel vaccine with high immune efficacy is urgently-needed. In this study, we examined the efficacy of subunit vaccine against TiLV through intramuscular injection using inulin acetate (InAc) nanoparticles encapsulation. The S2 protein (encoded by segment 2 gene of TiLV) was used as the antigen and the delivery system was named as InAc-S2. The toxicity analysis showed InAc-S2 had no significant influence on fishes growing and development. Meanwhile, we found InAc-S2 has a dual-release pattern (initial burst release and sustained release) in antigen release. Subsequently, tilapia was injected intramuscularly by different treatments (PBS, InAc, S2, and InAc-S2), and the immune protective effect induced by these treatments was analyzed. We found a powerful and long-lasting humoral or cellular immune response (serum antibody production, immune-related parameters and immune-related genes expression) can be induced in fishes by injected with InAc-S2 in comparison with those injected with S2 antigen alone. Moreover, a relative higher survival rate was observed in fish vaccinated with InAc-S2 at the dose of 4 μg protein/g of fish (with the relative percentage survival of 58.6%). Overall, this study for the first time demonstrated the potential of InAc as subunit vaccine carrier against TiLV infection and provide a reference to novel vaccine development in aquaculture.