BACKGROUND: Targeting CD20 antigen on B-lymphocytes provides good opportunity for management of the target cells in patients with B-cell malignancies. By the advent of hybridoma technology, monoclonal antibodies applications exert extensive changes in medical fields such as diagnosis, treatment and purification. OBJECTIVE: The prim aim of this study was to produce monoclonal antibody against CD20 for exploitation in diagnosis. METHODS: In this study, Balb/c mice were immunized with two peptides from extracellular domain of CD20. Poly Ethylene Glycol (PEG) fused spleen cells of the most immune mouse with SP2/0 (myeloma cells). Supernatant of hybridoma cells were screened for detection of antibody by ELISA. The desired clones were selected for limiting dilution (L.D). Afterward, specificity and cross reactivity of these antibodies were determined by immunological assay such as ELISA and western blot analysis (WB) and Immunofluorescence. Large scale of monoclonal antibodies was produced by ascetic fluid method. Monoclonal antibody was purified by chromatography then confirmed by SDS-PAGE. RESULTS: In this study, between five positive clone wells, 3 clones were chosen for limiting dilution. Limiting dilution product was one monoclone with absorbance about 2. CONCLUSIONS: These results indicate that such monoclonal antibodies against CD20 can be used in diagnosis of CD20 in the cells surface.