In order to construct the yeast display system of the human proteasome subunit alpha 6 (α6) and obtain its specific monoclonal antibodies for epitope analysis and mechanism investigation of ubiquitin-proteasome pathway, and set up a new rapid efficient way for the preparation of specific monoclonal antibodies (MAbs) without proteantigens which applicated recombinant antigen into detection directly, the gene PSA6_HUMAN coding human proteasome subunit alpha 6 was cloned into a yeast-displaying expression vector, pICAS-H, which had been inserted a His.tag marker for expression level detection. As probed with a His.tag monoclonal antibody and a specific monoclonal antibody generated by hybridoma, a recombinant yeast strain, α6-MT8, was selected by flow cytometry and fluorescence microscopy analysis. Combining with enzyme-linked immunosorbent assay (ELISA), ‘yeast-ELISA’ detection was established by basing on Saccharomyces cerevisiae cell surface engineering and applied to examined monoclonal antibody and its valance. The yeast-displaying recombinant antigen α6 with highly specific affinity was expressed efficiently after 48 h cultivation. The ‘yeast-ELISA’ was demonstrated primarily to detect and screen monoclonal antibody successfully.