Monoclonal antibodies or specific antibodies are now an essential tool of biomedical research and are of great commercial and medical value. The purpose of this study was to produce large scale of monoclonal antibody against CD34 in order to diagnostic application in leukemia and purification of human hematopoietic stem/progenitor cells. For large scale production of monoclonal antibody, hybridoma cells that produce monoclonal antibody against human CD34 were injected into the peritoneum of the Balb/c mice which have previously been primed with 0.5 ml pristane. Approximately, 5 ml ascitic fluid was harvested from each mouse. Evaluation of mAb titration was assessed by ELISA method. MAb was purified from ascitic fluid by affinity chromatography on protein A-Sepharose and then, the purity of mAb was assessed by SDS-PAGE and conjugated with FITC. Specific binding of conjugated mAb with CD34 marker of human hematopoietic stem/progenitor cells was evaluated using flowcytometry and immunofloresance techniques. This mAb showed appropriate reactivity in the following assays as if ELISA, Flowcytometry, Immunofluorescence staining and Western blot (WB). The results of immunological assays clearly showed that our antibody strongly reacts with CD34 antigen. The conjugated monoclonal antibody could be a useful tool for isolation, purification of human hematopoietic stem cells.