Despite effective vaccination programs in many countries, HBV infection is still a serious health problem throughout the world; more than 2 billion people have been infected with Hepatitis B virus (HBV). The serologic markers are crucial indicators in clinical diagnosis of HBV infection. The persistent presence of anti-HBc is associated with chronicity, and anti-HBe is an indicator for active viral replication. In the present study, two different hybridoma clones, 12E5 and 16F8, secreting anti-HBeAg and anti-HBc antibody were developed using hybridoma technology. BALB/c mice were immunized with HBe antigen (HBeAg), and monoclonal antibodies were generated from the spleen and lymph nodes of mice. Immunoglobulin types of antibodies were found to be IgG2a and IgG1, respectively. Monoclonal antibodies (MAbs) were produced in large scale, purified with affinity chromatography, and epitope analysis was performed. The results have shown that 12E5 and 16F8 monoclonal antibodies can be used for detection of HBcAg and HBeAg, indicating that they have the potential for use in clinical diagnosis.