Poly (I:C) can work as an immunostimulant and a viral vaccine; however, its functional mechanism in aquatic animals needs to be further investigated. In this study, comparative transcriptomic analyses were performed to investigate the effects of poly (I:C) on Argyrosomus japonicus at 12 h and 48 h postinjection. A total of 194 and 294 differentially expressed genes were obtained in the liver and spleen, respectively. At 12 h, poly (I:C) injection could significantly influence the function of the metabolism-related pathways and immune-related pathways in the liver through the upregulation of the genes GST, LPIN, FOXO1, CYP24A1, ECM1, and SGK1, and the downregulation of the genes IL-1β, CXC19, TNFAIP3, and IRF1. At 48 h, poly (I:C) could enhance the liver energy metabolism by upregulating the genes TXNRD and ECM1, while it also induced some injury in the cells with the downregulation of the genes HBA and CYP24A1. In the spleen, poly (I:C) could regulate the fish immunity and inflammatory response by upregulating the genes DDIT4, C3, EFNA, and MNK, and by downregulating the genes ABCA1, SORT1, TNF, TLR2, IL8, and MHCII at 12 h, and at 48 h, the poly (I:C) had a similar influence as that in the liver. Intersection analyses demonstrated that CYP24A1 and ECM1 were the main functional genes that contributed to the health of the liver. Ten and four genes participated in maintaining the health of the two tissues after 12 h and 48 h, respectively. In summary, our results provided a new insight into ploy (I:C) application in A. japonicus, and it also helped us to better understand the fish response mechanism to the viral vaccine injection.