Objective: To prepare and characterize monoclonal antibody against tumor-associated antigen OVA66, so as to provide effective method for further studying the biological function of OVA66. Methods: Recombinant pET32b-OVA66 plasmid was constructed and transfected into E. coli BL21 (DE3). His-OVA66 fusion protein was induced by IPTG and purified through Ni-TED affinity chromatography. Hybridoma cells stably secreting anti-OVA66 monoclonal antibodies were prepared by hybridoma technology. Biological and immunologic properties of monoclonal antibodies were determined by ELISA and Western blotting. The prepared antibody was applied in immunofluorescence, flow cytometry and immunohistochemistry procedures. Results: Recombinant pET32b-OVA66 plasmid was successfully constructed, and OVA66 fusion protein was induced and further purified. Two hybridoma cell strains secreting OVA66 monoclonal antibodies were established by hybridoma technology and named as 5F4 and 4G9, respectively. These monoclonal antibodies were characterized as IgG1 subclass and kappa subtype with affinity constant ka being 2.96 × 10 and 0.4 × 10 L/mol, respectively. Preliminary data showed that these antibodies acted against different antigenic epitopes. These antibodies could be used for the detection of OVA66 expression in immunofluorescence and flow cytometry assay, whereas 5F4 could also be used in immunohistochemistry assay. Conclusion: Two hybridoma cell strains secreting monoclonal antibodies against OVA66 have been successfully established, which facilitates further studies on biological functions of OVA66 and its potential in clinical application.