Chronic hepatitis B (CHB) infection remains worldwide health problem. Antiviral treatment options for CHB patients include nucleos(t)ide analogs (NAs) and interferon. Most of the current biomarkers for predicting treatment response are virus-dependent. MicroRNA-122 is the most abundant liver-specific miRNA and has been identified involved in multiple liver physiology and pathology including hepatotropic virus infection. To identify the role of miR-122 in NA therapy, 80 CHB patients with high viral load (HVL) were enrolled and serum miR-122 levels at baseline, week 12 and week 24 were measured. Serum miR-122 levels were significantly lower in patients who developed virological response (VR), compared with non-VR group. Levels of miR-122 at week 12 and week 24 were determined to be independent prognostic indicators for a VR with satisfactory AUROC values at 0.812 and 0.800, respectively. During NA therapy, serum miR-122 level deceased steadily and an earlier reduction was observed in VR group, indicating early reduction of miR-122 level might increase the possibility of developing virological response. In conclusion, we identified the dynamic change of serum miR-122 level and miR-122 levels at week 12 and week 24 as independent predictors for VR in CHB patients with HVL treated with NAs.