Objective:To construct a single chain MHC-epitope tetramers for detecting epitope specific cytotoxic T lymphocyte. Motheds:The C terminus of β2m fusing with extracellular domains of HLA A0201 was connected with a accessional BirA substrate peptide by a linker through restriction enzyme digestion and ligation. Results:Expression of the recombinant in a pET systerm as inclusion body was followed by refolded in the presence of the antigenic peptide. After biotinylization of the MHC-peptide complex, the tetramers was obtained by the coupling of a fluorchrome conjugated streptavidin with the complex. Conclusion:The novel method of constitution of tetramers was more efficient than the old labor-consuming one.