Abstract 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 (Gly 4Ser) 3 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 1 l 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.