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Major histocompatibility complex (MHC) class Ⅰ tetramer technology has become the central technique for analyzing antigen-specific CD8+ T cell responses and it has been widely used to explore the differentiation and formation of memory CD8+ T cells. Previously, a simplified and efficient procedure for preparing high quality HLA-A*0201 tetramers has been established in our lab and the tetramers loaded with HCMV peptide pp65495-503has been successfully applied to investigate HCMV-specific CD8+ T cells in Chinese populations. Using similar procedure we reported here the construction of HLA-A*0201 tetramer loaded with another dominant epitope derived from immediate early (IE)-1316-324 (VLEETSVML, VLE) of HCMV (A2-VLE) and characterization of this tetramer. After A2-VLE monomer was prepared and purified, its tetramer was then formed at a yield of 83%. The optimized amount of A2-VLE tetramer for staining 100 μl whole blood was 0.5 μg with incubation at 4℃ for 1 h.Furthermore, the dissociation constant of the tetramer binding to the specific CD8+ T cells of one HLA-A2+ donor was estimated to be 32.7 nmol/L, which is markedly higher than that of MHC monomer. The construction of A2-VLE tetramer provides an alternative choice for investigating HCMV-specific CD8+ T cell responses and will deepen our understanding of the differentiation and formation of HCMV-specific memory CD8+ T cells.