Abstract: To study the transmembrane activity of Heparin Binding Domain（HBD） derived from human fused at N terminal of heterologous protein, the expression plasmid pET28b HBD EGFP Histag was constructed by genetically fusing HBD to the N terminal of enhanced green fluorescent protein（EGFP）. The fusion protein HBD EGFP was expressed in E.coli and purified by Ni2+ NTA affinity chromatography. The results show that green fluorescence can be observed in HeLa cells using laser scanning confocal microscope when HeLa cells were co cultured with HBD EGFP. Flow cytometry results show that the transmembrane efficiency of HBD fused at the N terminal of EGFP is improved about 10 times compared with the HBD fused at the C terminal of EGFP. These results demonstrated that HBD fused to N terminal of heteologous protein exhibited tansmembrane activity. The HBD fused at the N terminal of EGFP had a higher efficiency than the HBD fused at the C terminal of EGFP. These results will provide theoretical foundation for the efficient application of HBD in the field of the delivery of anticancer drugs.