Interferon regulators (irf3), a member of the interferon regulator (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 (O. interferon regulatory factors 3, Opirf3) comes from the laboratory O. punctatus genome database, and the CDS sequence of Opirf3 has a total length of 1362 bp, can encode 453 amino acids, predicts its protein molecular weight of 50.0 kDa, 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 and kidney tissues of O. punctatus. When SKIV-SD infection was infected for 7 days, the expression level of Opirf3 in the liver, spleen and kidney of immune tissues was significantly regulated. In vitro stimulation experiments of the kidney cell line of O. punctatus showed that the expression of Opirf3 was significantly higher than that of the control group after 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 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. The above results may indicate that the Opirf3 gene is involved in the innate immune response of type I IFN in the anti-SKIV-SD of O. punctatus. This study can provide a theoretical basis for the molecular breeding of disease resistance in O. punctatus.