Interferon regulatory factor 3, a member of the interferon regulator factor (IRF) family, are major transcriptional regulators of type I interferon-dependent immune responses and play an important role in innate immune responses against DNA and RNA viruses. In this study, The irf3 gene (interferon regulatory factors 3, Opirf3) was obtained from the Oplegnathus punctatus genome database, and analyses found that the CDS sequence of Opirf3 has a total length of 1 362 bp, encoding 453 amino acids. The molecular weight of predicted protein is 50.0 ku, the theoretical isoelectric point is 4.97, and has an IRF domain and an IRF-3 domain. RT-PCR showed that Opirf3 was expressed in liver, gills, heart, skin, spleen, intestine, brain, kidney, stomach and head kidney of O. punctatus. When the fish were infected with SKIV-SD for 7 days, the expression levels of Opirf3 in the liver, spleen and kidney of immune tissues were significantly upregulated. In vitro stimulation experiments of the kidney cell line of O. punctatus showed that the expression of Opirf3 was significantly upregulated after stimulation with different concentrations of poly I:C, and the relative expression level of Opirf3 was the highest at a concentration of 100 μg/mL, which was 87.9 times higher than that of the control group. After siRNA interference, the expression level of Opirf3 was significantly reduced by 30%, the Opirf3 related genes IFN-α, CD40, CD80 and IL-1β were significantly downregulated, and IL-6 was significantly upregulated. These results may indicate that the Opirf3 gene is involved in the innate immune response of type I IFN in the resistance to SKIV-SD of O. punctatus. This study can provide a theoretical basis for the molecular breeding of disease resistance of O. punctatus.