A successful viral infection is primarily dependent on interactions between the viral pathogenesis and the host's antiviral immunity. MicroRNAs (miRNAs) are non-coding regulatory RNAs that play significant roles in modulating the host's antiviral immune responses. To elucidate the functional mechanism of miRNAs during megalocytivirus infection, in this study, micro-transcriptome analysis was performed on the head-kidney of turbot (Scophthalmus maximus) infected with RBIV-C1 or not at 3, 6 and 9 days after infection. A total of 247 differentially expressed miRNAs (DEmiRs) were identified, among which 125 were known miRNA and 122 were newly identified. Based on an integrated negative correlation analysis of miRNA-mRNA, a total of 1173 up-regulated and 644 down-regulated target genes (DEmRs) were identified. These DEmRs were found to be enriched in different KEGG pathways associated with signal transduction, endocrine system, immune system, cancer, and infectious diseases. Moreover, networks of immune-related DEmiRs-DEmRs were constructed and analyzed, revealing significant associations between RBIV-C1 infection and immune-related pathways, such as pattern recognition receptor, apoptosis, phagolysosome, autophagy, TNF signaling, adaptive immunity, cytotoxicity, and NF-kappa B signaling. Additionally, the negative correlation network analysis unveiled numerous known and novel DEmiRs that directly target RBIV-C1 genes. This study presents, for the first time, a comprehensive micro-transcriptome dataset of turbot following RBIV-C1 infection, offering valuable insights into the underlying survival mechanisms that involve immune evasion and antiviral immunity.