The acute‐on‐chronic liver failure (AoCLF) caused by hepatitis B virus (HBV) infection remains to be a challenge in clinics with a high mortality rate in China, and it is important to identify biomarkers to foresee the prognosis of patients with HBV. The current study analysed serum proteome changes of acute‐on‐chronic liver failure as a result of acute exacerbation of chronic hepatitis B infection. Serum samples were collected from normal subjects (NS, n = 8), patients with chronic hepatitis B (CHB, n = 12) and patients with AoCLF (n = 12). After removal of albumin/IgG and ultramembrane centrifugation, serum proteins were separated by two‐dimensional gel electrophoresis. Differentially expressed spots were identified by matrix‐associated laser desorption ionization time‐of‐flight tandem mass spectrometry. Through the removal of albumin/IgG and ultramembrane centrifugation, the well‐resolved and reproducible two‐dimensional gel electrophoresis (2‐DE) profiles were obtained. A total of 23 proteins were identified on 2‐DE profiles by their differential expression between the three cohorts. Mass spectrometry analysis resulted in the identification of 12 proteins unambiguously. Western blot analysis confirmed the proteomics results that the α1‐acid glycoprotein (α1‐AGP) levels decrease significantly in plasma of patients with AoCLF, but somewhat decreased in patients with chronic HBV. Further α1‐AGP levels in bulk serum samples were measured by immune turbidimetry including normal subjects group (n = 25), acute hepatitis group (n = 36), chronic hepatitis group (n = 52) and AoCLF group (n = 48), the level of α1‐AGP in AoCLF groups sharply decrease than other groups. Our study shows that α1‐AGP may be a potential plasma biomarker for AoCLF diagnosis because of acute exacerbation of chronic hepatitis B infection.