As a co-stimulatory signaling molecule, CD2 provides a secondary stimulatory signal during T-cell activation. Research on CD2 in lower vertebrates remains limited. In this article, we identify the tilapia CD2 gene (GenBank accession number: OM974656
designated as OnCD2), which is predominantly expressed in various immune tissues of tilapia. Additionally, we determine the interaction between tilapia CD2 and its ligand, CD48. Using the magnetic bead method, we identified the CD2-positive lymphocyte population in tilapia, which primarily includes Th, NCC, and B cells. Under different stimuli, the proportion of CD2-positive cells in the head kidney, peripheral blood, and spleen lymphocytes exhibited varying degrees of increase. Incubation of tilapia lymphocytes with CD2 antibody and CD48 recombinant protein upregulated the expression of genes associated with T cell activation. The results of this study demonstrate that tilapia CD2 not only plays a role in T cell activation through its interaction with the ligand CD48, but also mediates a more complex immune regulation pathway in tilapia. This research serves as a reference for the classification of fish immune cells and enhances our understanding of T cell immunity in fish.