Schizothorax prenanti (S. prenanti) is an important economical cold-water fish species in southwestern China, but it is susceptible to various pathogens infection. In order to clearly elucidate the antiviral mechanism, in this study, we have analyzed the transcriptome of S. prenanti spleen after challenge with the virus mimic, poly (I:C) (pIC), using next generation sequencing technology (RNA-seq). A total of 313 differential expressed genes (DEGs) in spleen at 12 h were obtained after pIC treatment, including 268 significantly up-regulated unigenes (fold change > 2) and 45 significantly down-regulated unigenes (fold change > 2). Through the immune-related DEGs (IRDs) screening, 47 IRDs were used to establish heat map, which intuitively showed a significantly difference after pIC treatment. To validate the RNA-seq data and observe gene expression, the expression levels of 14 IRDs were detected by qPCR after pIC treatment at 0, 4, 8, 12, and 24 h. The results indicated that the qPCR data presented a positive line correlation with RNA-seq data, and the 14 IRDs were responsive to pIC stimulation except IL-1β. Thus, based on the RNA-seq and qPCR data, we inferred that MDA5- and Jak-mediated signaling pathways may involve in the antiviral signaling transduction, and induce type I IFNs and ISGs to block virus invasion, respectively. Unfortunately, TLR3 and TLR22, as receptors of virus dsRNA, were no significantly expressed in this study. Nonetheless, our study still provides useful mRNA sequences of antiviral immunity for further immunological research, and facilitates improving disease restriction in S. prenanti.