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CD28 and CD80/86 are crucial co-stimulatory molecules for the T cell activation. Previous study illustrated that CD28 and CD80/86 present on T cells and antigen-presenting cells in flounder (Paralichthys olivaceus), respectively. The co-stimulatory molecules were closely associated with cell immunity. In this paper, recombinant protein of flounder CD80/86 (rCD80/86) and phytohemagglutinin (PHA) were added to peripheral blood leukocytes (PBLs) in vitro. Lymphocytes were significantly proliferated with CFSE staining, and the proportion of CD4+ and CD28+ lymphocytes significantly increased. In the meantime, genes related to the CD28-CD80/86 signaling pathway or T cell markers were significantly upregulated (p < 0.05). For further study, the interaction between CD80/86 and CD28 was confirmed. The plasmid of CD28 (pCD28-FLAG and pVN-CD28) or CD80/86 (pVC-CD80/86) was successfully constructed. In addition, pVN-ΔCD28 without the conserved motif "TFPPPF" was constructed. The results showed that bands of pCD28-FLAG bound to rCD80/86 were detected by both anti-FLAG and anti-CD80/86. pVN-CD28 complemented to pVC-CD80/86 showing positive fluorescent signals, and pVN-ΔCD28 failed to combine with pVC-CD80/86. The motif "TFPPPF" in CD28 played a crucial role in this linkage. These results indicate that CD28 and CD80/86 molecules interact with each other, and their binding may modulate T lymphocytes immune response in flounder. This study proved the existence of CD28-CD80/86 signaling pathway in flounder. • RCD80/86 and PHA induced lymphocyte proliferation and proportion of CD4+ cells in flounder. • RCD80/86 and PHA upregulated the expression of B7-CD28 signaling pathway genes, T cell markers, and cytokines. • PCD28-FLAG bound to rCD80/86, and pVN-CD28 complemented to pVC-CD80/86 in vitro. • Motif of "TFPPPF" in CD28 plays crucial role in the interaction between CD28 and CD80/86. [ABSTRACT FROM AUTHOR]