HBV-miR-3 is encoded by HBV and takes part in pathogenesis of HBV-related liver disease. Whether HBV-miR-3 has a relationship with HBV replication and is predictive of PegIFN-α treatment response is still unknown. HBV-miR-3 quantification is based on qRT-PCR. The relationship of HBV-miR-3 and HBV replication, and predictive value of HBV-miR-3 were evaluated in a cohort of 650 HBeAg positive patients from a multi-center, randomized phase III clinical trial for the study of PegIFN-a2b. HBV-miR-3 is significantly positively related to HBVDNA, HBVpgRNA, HBeAg and HBsAg at baseline and at all the different time points during PegIFN-α treatment. Both univariate regression analyses and multivariate logistic regression analyses showed HBV-miR-3 is a predictor of HBeAg seroconversion in the patients treated with PegIFN-α at weeks of 0, 12, and 24. 70.0% of patients with HBV-miR-3 < 3log at week 12 achieved HBeAg seroconversion, otherwise, with HBV-miR-3 > 6log at week 12 no patient obtained HBeAg seroconversion. Conbination of HBV-miR-3 and HBeAg is more strongly predictive of HBeAg seroconversion (83.64%) at week 12. HBV-miR-3 is new biomarker for HBV replication and positively correlated to HBV replication. HBV-miR-3 is also an early predictor of HBeAg seroconversion in the patients treated with PegIFN-α.