Objective  To express recombinant human MHCⅠchain related protein A α3 domain (MICA-α3) and natural killer group 2D (NKG2D), prepare anti-MICA-α3 monoclonal antibodies and test antibody binding function. Methods  MICA-α3 and NKG2D expression vectors MICA-α3-pET11c and NKG2D-pET16b were constructed and transformed into BL21 for expression. Recombinant proteins were purified by polyhistidine-tag chromatography and gel chromatography. After immunizing mice with MICA-α3 protein and screening monoclonal antibody by hybridoma technology, the monoclonal antibody function was tested by cell-ELISA. Results  Recombinant proteins were expressed in the form of inclusion body in E.coli  BL21. Five monoclonal antibodys were screened, which could bind to MDA-MB-453 cells espressing MICA-α3，and this binding did not affect the binding of MDA-MB-453 cells to NKG2D. Conclusion  Anti MICA-α3 monoclonal antibody can bind to tumor cells without affecting the binding of tumor cells to NKG2D.