Objective：To study interaction sites of the glucose-regulated protein 78 kD（GRP78） with pre-S1 protein（PreS1） of hep－atitis B virus（HBV）. Methods：Being amplified by PCR，the open reading frame of GRP78 was subcloned into pW28 and expressed in Escherichia coli（E.coli） B834. The GRP78 protein was purified by nickel affinity chromatography column. Three truncated plasmids of HBV PreS1，including pGST-PreS1-X1，pGST-PreS1-X2，pGST-PreS1-X3，were expressed in B834 and then the bacterial protein was purified by GST affinity chromatography column. Interactions between GRP78 and PreS1 truncations were detected by pull down and microscale thermophoresis（MST）. Results：The recombinant plasmid of pW28-GRP78 was successfully constructed. The GRP78 protein and the fusion protein（GST-PreS1-X1，GST-PreS1-X2，GST-PreS1-X3） were obtained. The directly interaction between GRP78 and fusion protein were detected by pull down and MST，suggesting that the 1-65 amino acid residues of PreS1 play a key role for GRP78 binding to PreS1. Conclusion：Thanks to the molecular cloning and purified technology of protein purification，the fu－sion protein of GRP78 protein and the PreS1 truncated seg－ments is formed. And interaction sites of GRP78 protein and the PreS1 is primarily compared，providing preliminary data for future study.