BACKGROUND Monoclonal antibody against alkaline phosphatase (Alp) has many applications which Alp-anti Alp complex (APAAP) formation is one of the most important ones. This complex is applicable in many immunohistochemical and immunocytochemical techniques such as diagnosis of various kinds of leukemias, lymphomas, skin diseases, kidney dysfunctions, etc. OBJECTIVE Production of anti-Alp monoclonal antibody for utilization in APAAP complex. METHODS After several arranged injections of Alp to Balb/c mice and determining the specific antibody titer by ELISA test, the spleen lymphocytes of immunized mice and Sp2/0 myeloma cells were fused using polyethylene glycol as fusing agent and hybridoma cells were cultured in HAT medium. Identification and selection of anti-Alp producing clones were done by performing ELISA test on supernatants of all resulting clones. Limiting dilution method was used to attain monoclones and the effect of obtained antibodies on enzyme activity was investigated by a specific ELISA test. For production of concentrated Ab, the hybridoma cells were injected to peritoneal cavity of mice and the produced ascetic fluids were collected. Finally class and subclass of the obtained antibodies were determined by Isostrip kit. RESULTS After six rounds of fusion, 104 Hybridoma clones were obtained and two Anti-Alp producing clones (A_1G_8 and A_1G_9) were selected and subcloned. Both antibodies were IgG_1 with kappa (kappa) light chains. These antibodies did not affect the enzyme activity and the electrophoresis of ascetic fluids showed an obvious band in gamma (gamma) position. CONCLUSIONS Because these antibodies are from IgG class and don't affect the enzyme activity, it seems that they are suitable for APAAP complex formation.