Abstract: Grass carp (Ctenopharyngodon idellus) which has importantly economic value is the most cultured freshwater fish in China, even in the world. The hemorrhagic disease caused by grass carp reovirus (GCRV) has greatly affected the sustainable development of grass carp aquaculture. GCRV vaccine is an important method to prevent and control hemorrhagic disease, but the multi-subtype and variation characteristics of GCRV limit the preventive effect of GCRV vaccine. Organismal immune ability is closely related to genetic factors, so many progresses have been made in the exploration of disease resistance breeding, such as molecular marker-assisted breeding. miRNAs are a class of about 22 nt in length, endogenous, evolutionarily highly conserved non-coding small RNA single-stranded molecules, regulate various biological activities of organisms by inhibiting the translation of target genes or degrade target genes by binding to the 3′ untranslated regions (UTR). miRNAs are important candidate functional molecules for the study of disease resistance in fish and were verified involving in the innate immune process during virus infection in the antiviral immune response. So not only expressed genes, miRNAs are also expected to be list as major resistance molecular resources in the selective breeding of highly resistant grass carp. In this study, miRNA sequencing has been applied for screening candidate functional miRNA, a total of 821 mature miRNAs were identified by miRNA sequencing, of which 118 were specifically expressed after GCRV challenge group and 82 were specific in unchallenged group in the head kidney. Differential analysis of miRNAs showed that 88 miRNAs were significantly down-regulated and 46 miRNAs were significantly up-regulated after GCRV challenge. Differentially expressed miRNAs (DE miRNAs) target genes are significantly enriched, which showed that DNA transcriptional regulation, apoptosis regulation, phosphorylation, negative regulation of receptor signaling pathway through JAK-STAT pathway, and cytokine-regulated signaling pathway are closely related to immune regulation in GO enrichment analysis. At the same time, functional enrichment results of KEGG showed valine, leucine, and isoleucine degradation (ubiquitin-mediated proteolysis), TGF-β signaling pathway, RIG-I receptor signaling pathway, PPAR signaling pathway, and p53 signaling pathway (p53 signaling pathway) pathway, FOXO and other immune-related signaling pathways were significantly enriched, target genes in these immune-related pathways are worthy further study. qPCR detection verified that the accuracy of sequencing and indicated that the expression level of miR-21 was the highest in the head kidney of the GCRV-challenge group, and foldchange between two groups of miR-194a was as high as 48 times, suggesting that different miRNAs in grass carp play different potential functions. Tissue distribution results of the screened DE miRNAs showed that miR-200 family members were the highest in pituitary, followed by immune tissues such as kidney, skin and gill, miR-100, miR-21 and miR-722 was the highest in muscle, spleen and liver, respectively. miR-194a was the highest in intestinal tract, followed by liver, kidney; and miR-122 was the highest in liver, followed by spleen and skin. These results showed that different miRNAs function diversify in tissues in the GCRV response process. In all, this study used high-throughput sequencing to analyze the differences of miRNAs in the head kidney of grass carp after GCRV challenge, aiming to excavate the functional miRNAs related to the GCRV response and screen the candidate resistant functional miRNA molecules, which will provide theoretical supplement and molecular reference for the study of disease-related molecules of grass carp and provide molecular resources for the selective breeding of disease-resistant grass carp.