Objective: To modify heparanase gene core promoter with amplified SV40 enhancer DNA sequence and analyze its activity. Methods: SV40 enhancer sequence was amplified and inserted into the multiple clone site of pEGFP-HPSEp to construct a recombinant plasmids named pEGFP-HPSEp-SV40enh. The vector driven by HPSEp-SV40enh and HPSEp were transfected into human tumor cell lines including hepatoma carcinoma cell line(HepG2), laryngocarcinoma cell line(Hep2) and chronic myelogenous leukemia cell line(K562), respectively. The activity of reporter gene GFP was detected using fluorescence microscope and flow cytometry after transfection. Results: The length of amplified SV40 enhancer was 237 bp and the sequence was accordant with the GeneBank data. The enzyme result of digestion and sequencing of the constructed vector pEGFP-HPSEp-SV40enh was consistent with the expectation, SV40 enhancer sequence was inserted into the allocated multiple clone site. Fluorimetric analysis revealed that hyperfluorescence was found in pEGFP-HPSEp-SV40enh group and less fluorescence was found in pEGFP-HPSEp group,The average transfection efficiencies of GFP-HPSEp-SV40enh in HepG2, Hep2 and K562 cells were 3.7%, 6.0% and 5.5%, respectively, while those of pEGFP-HPSEp were 4.8%, 13.4% and 7.7%, respectively, with all the ratios of them less than 1. Conclusions: SV40 enhancer sequence is amplified successfully and pEGFP-HPSEp-SV40enh could be successfully constructed, SV40 enhancer might improve the activity of HPSE promoter.