Antiviral immune responses are triggered by the innate immune recognition of viral infection. Toll/interleukin-1 receptor (TIR) domain containing adapter inducing interferon-β (TRIF) is an adapter in responding to activation of Toll-like receptors, which provides early clearance of viral pathogens. Our study focuses on the functional characterization of grouper TRIF (EcTRIF) based on the comparison of its sequence and functional evolution from grouper fish to mammals. The results show that the open reading frame of EcTRIF encoded a protein of 580 amino acids. Real-time PCR analysis indicates that EcTRIF was constitutively expressed in all the analyzed tissues in healthy grouper. EcTRIF was significantly induced in spleen post-LPS and poly (I:C) stimulation. Fluorescence microscopy shows that EcTRIF is colocalized with a Golgi apparatus marker, implying its unique subcellular localization in the Golgi apparatus. Luciferase reporter assays confirmed that EcTRIF was able to activate the IFN and NF-κB promoter. Overexpression of EcTRIF in grouper brain cells inhibited the replication of red-spotted grouper nervous necrosis virus (RGNNV). These results indicate that EcTRIF plays an important role in modulating antiviral innate immune responses. Our results have applications in functional studies on TRIF in teleost fish and immune evolution.