Abstract Background Hepatitis B virus (HBV) infection is a serious health problem worldwide. Treatment recommendation and response are mainly indicated by viral load, e antigen (HBeAg) seroconversion, and ALT levels. The S antigen (HBsAg) seroconversion is much less frequent. Since HBeAg can be negative in the presence of high viral replication, preS antigen (HBpreSAg) might be a useful indicator in management of chronic HBV infection. Results A new assay of double antibody sandwich ELISA was established to detect preS antigens. Sera of 104 HBeAg-negative and 50 HBeAg-positive chronic hepatitis B patients have been studied and 23 HBeAg-positive patients were enrolled in a treatment follow-up study. 70% of the HBeAg-positive patients and 47% of the HBeAg-negative patients showed HBpreSAg positive. Particularly, in the HBeAg-negative patients, 30 out of 47 HBpreSAg positive patients showed no evidence of viral replication based on HBV DNA copies. A comparison with HBV DNA copies demonstrated that the overall accuracy of the HBpreSAg test could reach 72% for active HBV replication. HBpreSAg changes were well correlated with changes of HBsAg, HBV DNA and ALT levels during the course of IFN-α treatment and follow-up. HBeAg positive patients responded well to treatment when reduction of HBpreSAg levels was more pronounced. Conclusion Our results suggested that HBpreSAg could be detected effectively, and well correlated with HBsAg and HBV DNA copies. The reduction of HBpreSAg levels in conjunction with the HBV DNA copies appears to be an improved predictor of treatment outcome.