Tumour necrosis factor (TNF) is one of the most pivotal factors of the TNF family and plays an essential biological role in immunity. However, the antibacterial function and mechanism of TNFα in teleosts are relatively poorly understood. In this study, a novel TNFα from Trachinotus ovatus (TroTNFα) was characterized. TroTNFα is widely expressed in immune tissues and increased after Vibrio harveyi infection. The recombinant protein TroTNFα facilitated the proliferation and chemotaxis of T. ovatus head kidney lymphocytes, induced apoptosis in human hepatocellular carcinoma cells (HepG2), and enhanced NF-κB promoter activity, whereas mutants with altered conserved receptor binding sites (Phe and Tyr mutated to Ala) lost these functions. Similarly, in vivo research revealed that, compared with the control, TroTNFα overexpression significantly reduced bacterial colonization, whereas the bacterial colonization of the mutants was similar to that of the control. Furthermore, our results showed that TroTNFα increased the vaccine-induced immune responses induced by the DNA vaccine pCTssJ against V. harveyi. Taken together, our results indicate that TroTNFα plays an indispensable role in antibacterial immunity, providing the first evidence that the binding sites (Phe144 and Tyr216) of TroTNFα are crucial in these processes in teleosts and enhances DNA vaccine efficacy as an immune adjuvant.