Abstract The quantification of viral nucleic acids in serum by real-time PCR plays an important role in diagnosing hepatitis B virus and hepatitis C virus infection. In this study, we developed an assay using specific primers and probes to quantify hepatitis B virus DNA or hepatitis C virus RNA in serum from infected patients. For standardization and validation of the assay, an international panel of hepatitis B virus/hepatitis C virus and standard plasmids was used. A correlation coefficient of 0.983 and 0.963 for hepatitis B virus and hepatitis C virus, respectively, was obtained based on cycle threshold values and concentrations of DNA or RNA. The standard curve showed a linear relationship from 19 IU/mL to 1.9 × 109 IU/mL of serum, with a coefficient of determination (r2) of 0.99. In sera from patients infected with hepatitis B virus or hepatitis C virus viral loads (19 IU/mL and 1.9 × 109 IU/mL), we quantified viral loads with a detection limit of 1.9 × 102 IU/mL. The real-time quantitative PCR assay developed in this study provides an ideal system for routine diagnosis and confirmation of indeterminate serological results, especially in immunosuppressed patients.