Lateolabrax japonicus, a prevalent aquatic delicacy, is known to elicit allergic reactions in certain individuals. Nevertheless, the investigation into its allergenic components has remained notably inadequate. In the research, an approximately 35 kDa heat-stable protein of L. japonicus raw/steamed extracts was verified as tropomyosin (TM) by LC−MS/MS. Open reading frame of TM (852 bp) was acquired via PCR amplification, encoding 284 amino acids. The IgE-binding frequency of TM expressed in Escherichia coli was 22.5% among 80 fish-sensitized patients. Furthermore, TM had the ability to activate basophils in 7 patients. In the Balb/c mice model, compared with the PBS group, the levels of specific antibodies (IgE, IgG1, and IgG2a), CD19 + B cells, IL-4, and IL-10 were significantly increased in the TM group. However, the opposite was indeed the case for CD4 + TCR-β, CD4 + CD25 + Fox p 3 + cells, and IFN-γ. These findings regarding an allergen assist in conducting component-resolved diagnoses and therapeutic research for fish allergy.