Methodology for accurate quantification of intra-hepatic cccDNA has long been a technical challenge, yet it is highly desired in the clinic. Here, we developed a sensitive method for quantification of intrahepatic cccDNA in liver biopsies from patients, which allowed to predict patient's response to interferon therapy at baseline. Twenty-five patients with HBeAg+ CHB were recruited and liver biopsies were obtained at baseline and 1-year after interferon treatment, respectively. Both intrahepatic cccDNA and HBV DNA were absolutely quantified by a droplet digital PCR amplification system. Patients were categorized as either responder or non-responder group based on their HBeAg status 1-year after interferon therapy. Levels of both intrahepatic HBV DNA and HBV cccDNA were significantly reduced after interferon treatment among the responders, but not the non-responders, in comparison with their levels at baseline. Baseline values of intrahepatic HBV DNA over cccDNA significantly correlated with patient's response to PEG-IFN therapy (P = 0.000). In addition, HBeAg seroconversion also correlates with a significant reduction in intrahepatic pgRNA production among the responders after interferon therapy (P = 0.030). In conclusion, our results suggest that baseline value of intrahepatic HBV DNA over cccDNA may be a preferable indicator for selecting appropriate patients for IFN-based therapy in the clinic.