Hepatitis B virus (HBV) infection is a major health problem worldwide. The diagnosis of acute and chronic hepatitis B infection is based on the detection of hepatitis B surface antigen (HBsAg). We report here the development of hybrid cell producing monoclonal antibodies (MAbs) specific for HBsAg using hybridoma technology. BALB/c mice were immunized with a mixture of HBsAg subtype "ad" and subtype "ay." Spleen and lymph nodes were used as a source of high-titer antibody producing lymphocytes and removed and fused with myeloma cells of F0 origin separately. In the five fusion experiment, enzyme-linked immunosorbent assay (ELISA) tests showed that among 1594 hybridomas only 5 hybrids (9D12, 2B7, 4G5, 2G3, and 6E7) reacted with HBsAg. These MAbs were characterized for use in the development of diagnostic kits based on sandwich ELISA test system. The MAbs were conjugated with horseradish peroxidase (HRP) and used in the quick sandwich ELISA system. This system is a quite practical and time-saving test system when compared with common and commercial sandwich ELISA for diagnosis of hepatitis B surface antigen in human serum.