Objective: To investigate the screening of serum biomarkers in patients with HBV-related acute-on-chronic liver failure (HBV-ACLF) by isobaric tags for relative and absolute quantitation (iTRAQ). Methods: Gel electrophoresis was used to isolate and remove high-abundant proteins. Each group of peptides was labeled by the iTRAQ reagents and then tested with an UltiMateTM 3000 nanoliter high-performance liquid chromatograph, and a Q-Exactive tandem mass spectrometer. The Protein Discovery software was used to analyze mass spectrometry data and perform bioinformatic analysis for differentially expressed proteins. Results: Ten samples each were included in the HBV-ACLF group and the chronic hepatitis B (CHB) group, and six samples each were included in the HBV-ACLF survival group and the HBV-ACLF death group. Compared with the CHB group, the HBV-ACLF group had 43 differentially expressed proteins, among which 34 were downregulated and 9 were upregulated. Compared with the HBV-ACLF survival group, the HBV-ACLF death group had 33 differentially expressed proteins, among which 18 were upregulated and 15 were downregulated. Conclusion: Keratin,α1-acid glycoprotein, and zinc-α2-glycoprotein identified in the serum may be used as potential biomarkers for predicting the prognosis of patients with HBV-ACLF.