Singapore grouper iridovirus (SGIV), as a causative agent of serious systemic disease, resulted in significant economic losses in grouper aquaculture. In this study, recombinant eukaryotic vector pEGFP-ICP46 which was inserted with SGIV-ICP46 (infected cell polypeptides 46) gene was transfected into fathead minnow (FHM) cells, and ICP46-GFP fusion protein was successfully expressed in the cytoplasm of FHM cells. Candidate siRNA targeting SGIV-ICP46 gene (siRNA-ICP46) was designed and chemically synthesized. To investigate the inhibition effect of siRNAICP46, pEGFPICP46 and siRNA was co-transfected into FHM cells, and the green fluorescence was observed by fluorescence microscope at the indicated time points (24, 36, 48, 60 and 72 h post transfection). During 24-48 h after transfection, the green fluorescence in FHM cells co-transfected with siRNAICP46/pEGFPICP46 were similar to that of positive control (co-transfected with siRNAGFP/pEGFPICP46) , and both of them are about 70% weaker than that of negative control (co-transfected with siRNANegative / pEGFP-ICP46), which show the siRNAICP46 can effectively inhibit the extrinsic SGIV ICP46 gene in FHM cells during 24-48 h after transfection.