Purpose To investigate the predictive value of serum hepatitis B virus (HBV) RNA on HBeAg seroconversion in treated chronic hepatitis B (CHB) patients. Methods Sixty-four HBeAg-positive CHB patients were selected. They were divided into HBeAg seroconversion group including 11 cases and HBeAg non-seroconversion group including 53 cases. HBV RNA levels and other laboratory results were measured at baseline and week 12, 24, 48, 72 during treatment in both groups. The predictive value of HBV RNA level for the seroconversion of HBeAg in patients treated for hepatitis B was analyzed. Results Significant differences existed in serum HBV DNA and HBV RNA levels between the two groups at baseline while there was no significant difference in HBsAg. The correlation between HBV RNA and HBV DNA was significantly high ( r = 0.707, P < 0.05), while the correlation between HBV DNA and HBsAg ( r = 0.474, P < 0.05) or HBV RNA and HBsAg was poor ( r = 0.372, P < 0.05). Patients with younger age and higher HBV RNA levels at baseline and week 24 were less likely to have HBeAg seroconversion. HBV RNA was better than HBV DNA and HBsAg in predicting HBeAg seroconversion whether at baseline or week 12 and week 24. The area under the curve of HBV RNA level at 24th week was the highest, which was 0.942, and the cutoff value was 4.145 log 10 copies/ml. Conclusion HBV RNA level may be a suitable serum marker to predict whether HBeAg seroconversion can occur. CHB patients with serum HBV RNA level lower than 4.145 log 10 copies/ml at week 24 were more likely to achieve HBeAg seroconversion.