The principles governing gammadelta T cell specificity and diversity remain unclear due to lack of detailed structural analysis. To elucidate key structural basis of the specificity of gammadelta TCR for tumors, we analyzed the binding activities of synthesized TCR Vdelta2 CDR3 peptides derived from tumor infiltrating lymphocyte (TIL) s in ovarian epithelial carcinoma (OEC) via biospecific interaction analysis approach, enzyme immunoassay and immunofluorescence assays. Besides, we used human CDR3delta grafted-Ig to repeat major tests. We found that synthesized OEC-derived CDR3delta peptides could bind specifically to tumor cell lines and tissues. CDR3delta-graft Ig showed a similar binding specificity with CDR3delta peptides, suggesting the determinant role of CDR3delta in antigen binding. Moreover, CDR3delta peptide-mediated binding specificity was blocked by pre-incubation with same peptide, which decreased the cytotoxicity of gammadelta T cells to OEC cells in vitro. Our finding indicates that CDR3delta peptide could mimic antigen-binding specificity of gammadelta TCR. Our strategy provides a novel, simple and convenient approach to investigate the binding activity and function of gammadelta TCR.