PreS1 (21-47) region of HBV large surface protein is hepatocyte receptor binding site and the anti-preS1 (21-47) antibody possesses the virus-neutralizing activity and protective effect. It is important to obtain the peptide with higher immunoreactivity on a large scale for detecting the anti-preS1 (21-47) antibody in the sera from HBV infected patients and future vaccine recipients. The expression vector pGEX SLS, which expressed two copies of the preS1 (21-47) peptide connected by a flexible linker (Gly4Ser3) fused to glutathione S-transferase (GST), was constructed. The fusion protein, named GST-SLS, was highly expressed in E. coli and purified by affinity chromatography. Ninety milligrams purified protein can be obtained from 1l of culture. The data in ELISA analysis showed that the immunoreactivity of GST-SLS was enhanced significantly in comparison with GST-S II, a GST fusion protein with two copies preS1 (21-47) linked directly; GST-S I, another GST fusion protein with one copy preS1 (21-47) and preS1 (21-47) synthesized peptide. In addition, GST-SLS has been tried to use in detecting anti-preS1 (21-47) antibody in the sera from HBV infected patients and a satisfied result was gained. Therefore, GST-SLS may have potential to be developed into a new kit for diagnosis and prognosis of hepatitis B (HB) patients.