Toll/interleukin-1 receptor (TIR) domain containing adapter inducing interferon-β (TRIF) is an adapter in responding to activation of some toll-like receptors (TLRs), which provides early clearance of viral and bacterial pathogens. Here we identified and characterized a full-length genomic sequence of TRIF gene from grass carp Ctenopharyngodon idella (designated as CiTRIF). CiTRIF genomic sequence consists of 3534 base pairs (bp), containing 5’ flank sequence (496 bp) and unique intron (815 bp). The full-length cDNA sequence is 2241 bp, including 5’ untranslated region (UTR) of 352 bp, 3’ UTR of 209 bp, and an open reading frame of 1680 bp encoding a polypeptide of 559 amino acids with an estimated molecular weight of 62.643 kDa and a predicted isoelectric point of 5.71. The deduced amino acid sequence just contains TIR domain, and is most similar to the zebrafish (Danio rerio) TRIF sequence with an identity of 64%. CiTRIF exhibits sequence divergence from its orthologs. Promoter region was predicted and promoter activity was verified. mRNA expression of CiTRIF gene is widespread in fifteen tissues investigated, highly in foregut and skin physiological immune barrier. The transcripts of CiTRIF were significantly and rapidly induced in spleen and head kidney tissues at early stage post grass carp reovirus (GCRV) challenge. The modulations are significant but mild in CIK (C. idella kidney) cells post GCRV infection or poly(I:C) stimulation. The over-expression vector was constructed and transfected into CIK cell line to get stably expressing recombinant proteins. In CiTRIF transfected cells, mRNA expressions of CiTRIF, CiRIG-I, CiIRF7 and CiIFN-I were up-regulated. After GCRV infection, the transcripts of CiTRIF, CiRIG-I, CiIRF7 and CiIFN-I fell a little bit after a rapidly and strongly rise. In CiTRIF over-expression cells, virus load and titer were significantly lower than those in controls post GCRV challenge, and virus replication was inhibited obviously. The results indicate that the novel TRIF gene from grass carp plays important roles in modulating antiviral innate immune responses, and serve the further functional studies on TRIF gene in teleosts and immune evolution.