Hepatitis B surface antigen (HBsAg) levels are used to evaluate and monitor clinical phases of chronic hepatitis B infection but their clinical significance is unclear in the late complications, cirrhosis of the liver and hepatocellular carcinoma. This study aimed to evaluate HBsAg levels across the whole natural history of hepatitis B virus infection, including late complications. This retrospective, cross‐sectional study enrolled 838 treatment‐naive patients diagnosed with chronic hepatitis B infection at First Affiliated Hospital of Fujian Medical University between 2009 and 2012. Patients were classified into six groups: immunotolerance, immunoclearance, low replicative, negative hepatitis e (HBeAg) phases, liver cirrhosis, and hepatocellular carcinoma. Main outcome measures were serum HBsAg, HBeAg, HBV DNA, total bilirubin, albumin, alanine and aspartate aminotransferase, and quantitative correlation of HBsAg with HBV DNA. HBsAg levels declined significantly between clinical phases of infection (all P < 0.001) and were significantly lower in decompensated than in compensated cirrhosis (2.90 vs. 3.30, P < 0.001) but not significantly different between early versus advanced hepatocellular carcinoma. Significant positive correlations were observed between serum HBsAg and HBV DNA at immunoclearance and HBeAg negative phases, compensated and decompensated liver cirrhosis and advanced but not early hepatocellular carcinoma (all P < 0.001). HBsAg and HBV DNA were significantly higher in HBeAg positive patients with advanced hepatocellular carcinoma (P < 0.001). HBsAg levels differ significantly between chronic hepatitis B infection phases, decreasing progressively from chronic infection to cirrhosis and hepatocellular carcinoma. Significant correlations are found between serum HBsAg and HBV DNA. J. Med. Virol. 87:380–387, 2015. © 2014 Wiley Periodicals, Inc.