Quantification of cytotoxic T lymphocytes (CTL) is extremely important due to the pivotal role they play in controlling pathogen infection and anti-tumor actions. Previously used methods for detecting specific CTL are usually indirect. In recent years, tetramer technology has been developed to directly visualize antigen-specific CTL efficiently, and become the critical approach in studying T cell immune responses. A simplified procedure for preparing tetramers is reported here in this paper and a tetramer loaded with human cytomegalovirus (HCMV) peptide was successfully obtained using this procedure, which possessed binding activity with specific CTL. The heavy chain of HLA-A * 0201 gene was cloned by RT-PCR from HLA-A2+ donor. An expression vector, encoding the extracellular domain of HLA-A * 0201 heavy chain (A2) fused with a BirA substrate peptide (BSP) at its carboxyl terminus, was constructed by PCR with cloned A2 gene as the template. The A2 heavy chain was expressed in Escherichia coli mostly in the form of inclusion body and purified by washing inclusion body. The monomer of soluble A2 loaded with peptide was reconstructed by dilution from the heavy chain in the presence of light chain beta2-microglobulin and HLA-A2 restricted HCMV pp65(495-503) peptide (NLVPMVATV, NLV). Refolded A2-NLV monomer was biotinylated with a commercial BirA and purified by low pressure anion exchange chromatography on a Q-Sepharose (fast flow) column. The tetramer was then formed by mixing A2-NLV monomer with streptavidin-PE in a ratio of 4:0.8 leading to more than 85% multiplication as revealed by SDS-PADE under non-reducing conditions without boiling the sample. Flow cytometry analysis indicated that this tetramer could bind to specific CTL from HLA-A2+ donor. In conclusion, a simplified procedure is established to prepare HLA-A2 tetramer, which may not only facilitate the application of tetramer technology for studying specific T lymphocyte immune response but A2-NLV itself be applied clinically to monitor CMV-specific CTL in stem cell and organ transplantation.