To assess the significance of pre-S proteins expression during chronic hepatitis B virus (HBV) infection, we developed a sensitive labeled avidin biotin ELISA to detect pre-S1 and pre-S2 proteins. In serum specimens from 80 patients with chronic liver disease, the frequency of pre-S1 and S2 proteins was 53.7% and 47.5%, respectively. Furthermore, it reached 87.8% and 77.6%, respectively, in cases with chronic HBV infection, indicating that pre-S proteins are usually expressed in sera with chronic HBV infection. We found that the expression of pre-S proteins is closely associated with HBV replicating markers, such as HBV DNA, HBcAg and HBeAg, in sera of patients with chronic HBV infection. Both pre-S1 and S2 proteins were often concurrently expressed in liver and serum with chronic HBV infection. However, the frequency was slightly higher in liver than in serum, suggesting that it may be clinically valuable to detect pre-S proteins in serum and liver simultaneously to determine the status of HBV infection. Our results also indicated that pre-S proteins expression in serum can serve as markers of HBV infection, but cannot be used to estimate the severity and activity of hepatic pathology.