In order to improve the functional affinity of the humanized VH single domain antibody against human lung cancer, the genes coding the homogenous dimers dihu3D3VH and tetramers tehu3D3VH were constructed by fusing the SV5-Cys short peptide and p53 tetramerization structural domain gene to hu3D3VH gene via recombinant PCR technique, respectively. Then, the dihu3D3VH and tehu3D3VHgenes were cloned to the prokaryotic expression vector pET-22b ( + ) and expressed in E. coli BL21(DE3). The proteins expressed were purified through Ni2+ -affinity chromatographic column. Meanwhile,the hu3D3VH, dihu3D3VH and tehu3D3VH proteins were labeled with FITC, and their reactivity with antigen and specificity were analyzed by immunofluorescence assay. As to their functional affinities, it was analyzed and compared by flow cytometry. The results indicated that these two genes were expressed as monomers and mainly as inclusion bodies. After purification and renaturation, there were about 50% of dimers and 70% of tetramer remaining in the protein solution. In addition, the dihu3D3VH and tehu3D3VH proteins still remained the reactivity with antigen and specificity of hu3D3VH protein, and their functional affinities were increased about 60% or 100% respectively, compared with those of hu3D3VHprotein. It is evident that the functional affinity of hu3D3VH protein can be greatly improved by increasing its binding valency.