In this study, we evaluated the potential of poly (I:C) to induce antiviral status for protecting rock bream from RBIV infection. Rock bream injected with poly (I:C) at 2 days before infection (1.1 × 104) at 20 °C had significantly higher protection with RPS 13.4% and 33.4% at 100 and 200 μg/fish, respectively, through 100 days post infection (dpi). The addition of boost immunization with poly (I:C) at before/post infection at 20 °C clearly enhanced the level of protection showing 33.4% and 60.0% at 100 and 200 μg/fish, respectively. To investigate the development of a protective immune response, rock bream were re-infected with RBIV (1.1 × 107) at 200 dpi. While 100% of the previously unexposed fish died, 100% of the previously infected fish survived. Poly (I:C) induced TLR3 and Mx responses were observed at several sampling time points in the spleen, kidney and blood. Moreover, significantly high expression levels of IRF3 (2.9- and 3.1-fold at 1 d and 2 days post administration (dpa), respectively), ISG15 and PKR expression (5.4- and 10.2-fold at 2 dpa, respectively) were observed in the blood, but the expression levels were low in the spleen and kidney after poly (I:C) administration. Our results showed the induction of antiviral immune responses and indicate the possibility of developing long term preventive measures against RBIV using poly (I:C).