Prolonged infection of hepatitis B virus (HBV) is reported to cause hepatocellular carcinoma (HCC) via liver cirrhosis. However, it is still unknown whether the HCC is induced by the HBV DNA integration or by inflammatory stimulation during the phase of liver cirrhosis. The aim of this study is to determine the intracellular or intranuclear distribution of HBV DNA with a highly sensitive assay. Here we directly detected the integration of HBV DNA by fluorescence in situ polymerase chain reaction (FISPCR). Since FISPCR products directly incorporate rhodamine-4-dUTP, the nucleus of Alexander cells integrated with HBV gene reacted with the HBV primers emits obvious fluorescence. The fluorescence values which were measured with an imaging analyzer show a significant difference between Alexander cells as compared to the controls. In conclusion, the target sequences of HBV were specifically amplified as fluorescent DNA after the present FISPCR procedure. This method could provide a novel and simple strategy for determining the quantitative role of viral DNA integration in oncogenesis.