The cyclic GMP-AMP synthase (cGAS)-Stimulator of Interferon Genes (STING) pathway is a crucial component of innate immune system that protect host against various bacterial and viral pathogens. When cGAS senses foreign DNA or RNA in the cytoplasm of cells, it catalyzes the production of cGAMP, which can bind STING and activate the production of interferons (IFNs) and other cytokines that promote host defense. In this study, we identified a cGAS homologue (EacGAS) from red-spotted grouper. The open reading frame of EacGAS is 1509 bp in length, encoding a predicted protein of 502 amino acids. The domain prediction and phylogenetic analysis of EacGAS protein sequence was performed using online bioinformatics tools and software. To determine the spatial expression pattern of EacGAS, quantitative real-time PCR (qPCR) was conducted in 12 different tissues of the healthy, red-spotted groupers. The highest expression of EacGAS was found in the gill. Furthermore, the effects of immune stimulants on the temporal expression pattern of EacGAS was examined in the in the peripheral blood stem cells (PBSCs) of red-spotted groupers challenged with LPS, poly I:C, and nervous necrosis virus (NVV). Poly I:C challenge showed the highest induction effect on the expression of EacGAS at 24 h post-injection (h.p.i), while the LPS challenge stimulated the highest expression of EacGAS at 48 h.p.i. Under NNV challenge, the expression of EacGAS was gradually upregulated and reached peak at 48 h.pi. To further characterize the function of EacGAS, the expression of NF-κB was compared between the raw cells transfected with empty pcDNA3.1(+) vector and pcDNA3.1-EacGAS. The results revealed that the EacGAS overexpressing cells produced higher level of NF-κB, indicating NF-κB as a downstream mediator of cGAS-STING pathway in red-spotted grouper. The role of EacGAS in antiviral defense was investigated in the FHM cells transfected with pcDNA3.1-EacGAS. Compared to control cells, the EacGAS overexpressing cells showed significantly higher cell viability after VHSV infection. Taken together, our results suggest that EacGAS may act a key player in antiviral innate immune response via activation of NF-κB signal pathway in red-spotted grouper.