To develop a sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of serum hepatitis B virus large surface protein (HBV-LP), and study the clinical value of HBV-LP.Serum HBV-LP levels and a panel of other HBV markers were investigated in a large population of patients with chronic HBV. The clinical value of HBV-LP was evaluated by comparing the coincidence of detection of HBV markers and the change of serum HBV-LP level during antiviral therapy.The ELISA was found to be sensitive and specific for the detection of HBV-LP. Serum HBV-LP level was positively correlated with HBV DNA (r=0.743) in HBV patients. Among the five HBV markers tested, HBV-LP displayed the highest coincidence rate (94.7%) with HBV DNA.Serum HBV-LP was strongly correlated with HBV DNA. This ELISA therefore offers a promising approach for the diagnosis and treatment monitoring of HBV patients.