As an aquatic fish, the spotted halibut Verasper variegatus is highly susceptible to bacterial and virus infections. Tumor necrosis factor-alpha (TNF-α) as a cytokine could control the inflammatory responses. The functions of TNF-α in many species have been widely studied, particularly in mammals. However, little is known about the TNF-α functions in V . variegatus . We first cloned and sequenced the TNF-α gene in V . variegatus (VvTNF-α). The two conserved cysteine residues, transmembrane sequence, Thr-Leu motif, and TNF family signature, as well as the TA-rich motifs of its proteins related to inflammatory responses had high similarity to those of the other teleost and mammalian TNF-α. The phylogenetic analysis showed that VvTNF-α was consistent with TNF-α genes of other vertebrates. The VvTNF-α transcripts were extensively distributed in the peripheral blood leukocytes (PBLs), spleen, and gill, indicating that the VvTNF-α had a role in immune function. Furthermore, treatment with pathogen-associated molecular patts (PAMPs) could induce a rapid and significant increase of VvTNF-α in the PBLs, which reveals that VvTNF-α does participate in the host immune responses against bacterial and viral pathogens. We found that VvTNF-α had an interesting expression patt during metamorphosis, showing that the flatfish TNF-α may have some novel functions during specific developmental stages. In addition, the 3D structure prediction of VvTNF-α provided an indication of how it is likely to interact with other proteins. Therefore, VvTNF-α has multiple functions, and provides valuable information to explore novel functions of TNF-α.