Human Chorionic Gonadotropin (HCG) was purified from pregnant women urine. The HCG was extracted by ethanol precipitation and then purified fractions were obtained by gel filtration on sephacryl S300. B-HCG was then purified from preparated HCG by ion exchange chromatography on DEAE sepharose . Purified B-HCG was used to immunize BALB/C mice. Spleen cells from immunized mice were fused with P3X63/Ag8.653 myeloma cell line in the presence of polyethyleneglycol. Hybrids were propagated in RPMI - 1640 supplemented with Hypoxanthin, Aminoptren, and Thymidine (HAT). Monoclonal antibody was screened by using indirect enzyme linked immunosorbant assay (ELISA) to detect anti- B-HCG antibodies. Several clones were detected to produce B- HCG. One clone was selected and expended in tissue culture flask for further analysis of antibody specificity and cross reactivity with human leutinizing hormone (LH). Hybrid cell line was karyotyped and compared with myeloma cell line. The results demonstrated, the production of specific B-HCG monoclonal antibody, which does not cross react with LH.