Objective To prepare a monoclonal antibody (mAb) against human CD33 by immunizing mice with recombinant vector and analyze its characteristics and clinical application. Methods The eukaryotic expression vector pcDNA3.1(+)/CD33 was constructed and used to immunize mice. The mouse monoclonal antibody against human CD33 was then harvested using the hybridoma technique. Its properties were evaluated and the clinical performance was validated. Results One hybridoma cell line capable of secreting mouse anti-human CD33 monoclonal antibody was successfully obtained, which was named HI33a for clone identification with a subclass of IgG2a, κ. Flow Cytometry analysis revealed that the antibody could stain myeloid cell lines but not lymphoid cell lines, and it could inhibit the binding of similar imported antibodies with HL-60 cells competitively. Western blotting verified that it could bind a Mr 67 000 membrane protein extracted from HL-60 cells, which was a strong indication of the characteristics of CD33 protein molecule. Labeled with PE fluorescein, CD33-PE was tested as an antibody reagent in comparison with other similar imported products. Its overall performance including the accuracy, linearity, and precision all met the industrial standard. Further clinical evaluation of 558 bone marrow samples showed that the results were highly consistent with those by the imported reagents used as controls. Conclusion A hybridoma cell line stably secreting anti-human CD33 mAb was prepared.