To analyze the role of serum miR-125b-5p in reflecting liver damage and predicting outcomes in chronic hepatitis B (CHB) patients with acute-on-chronic liver failure (ACLF).CHB patients with normal hepatic function (n = 100), moderate-to-severe liver damage (n = 90), and ACLF (n = 136) were included. Among hepatitis B virus (HBV)-ACLF patients, 86 and 50 were in the training and validation cohorts, respectively. Serum miR-125b-5p level was measured by quantitative real-time PCR.Serum miR-125b-5p level increased with disease progression, and serum miR-125b-5p level was lower in surviving than in dead HBV-ACLF patients. Among HBV-ACLF patients, miR-125b-5p positively correlated with total bilirubin (TBil; r = 0.214, p < 0.05) and model for end-stage liver disease (MELD) score (r = 0.382, p < 0.001) and negatively correlated with prothrombin activity(PTA; r = -0.215, p < 0.05). MiR-122 showed a contrasting performance compared with miR-125b-5p. Cox regression analysis showed that miR-125b-5p, miR-122, and PTA were independent survival predictors for HBV-ACLF, and low miR-125b-5p and high miR-122 levels may predict a longer survival in HBV-ACLF. MiR-125b-5p (AUC = 0.814) had a higher performance for survival prediction in HBV-ACLF compared with miR-122 (AUC = 0.804), PTA (AUC = 0.762), MELD score (AUC = 0.799), and TBil (AUC = 0.670) alone; predictive effectiveness of miR-125b-5p was increased by combination with miR-122 (AUC = 0.898). MiR-125b-5p was an effective predictor of HBV-ACLF outcomes in the validation cohort.MiR-125b-5p increase is associated with severity of liver damage; high serum miR-125b-5p may serve as a predictor for poor outcomes in HBV-ACLF cases.