MHC/peptide tetramer technology has been widely used to study antigen-specific T cells, especially for identifying virus-specific CD8+ T cells in humans. The tetramer molecule is composed of HLA heavy chain, beta2-microglobulin (beta2m), an antigenic peptide, and fluorescent-labeled streptavidin. To further investigate the HLA-A*1101-restricted CD8+ T cell responses against human cytomegalovirus (HCMV), we established an approach to prepare HLA-A*1101 tetramer complexed with a peptide from HCMV. The cDNA encoding HLA-A*1101 heavy chain was cloned and the prokaryotic expression vector for the ectodomain of HLA-A*1101 fused with a BirA substrate peptide (HLA-A*1101-BSP) at its carboxyl terminus was constructed. The fusion protein was highly expressed as inclusion bodies under optimized conditions in Escherichia coli. Moreover, HLA-A*1101-BSP protein was refolded in the presence of beta2m and an HCMV peptide pp65(16-24) (GPISGHVLK, GPI). Soluble HLA-A*1101-GPI monomer was biotinylated and purified to a purity of 95%, which was subsequently combined with streptavidin to form tetramers at a yield of > 80%. The HLA-A*1101-GPI tetramers could bind to virus-specific CD8+ T cells, suggesting soluble HLA-A*1101-GPI tetramers were biologically functional. This study provides the basis for further evaluation of HLA-A*1101-restricted CD8+ T cell responses against HCMV infection.