Objective:To construct a EGFP(enhanced green fluorescent protein)-labled eukaryotic expression recombinant plasmid of heNOS(human endothelial nitric oxide synthase)gene. Methods:According to the gene of heNOS and the multiple clone site of the expression vector pEGFP-N1 plasmid,a specific pair of primers were designed and synthesized. PCR amplification of heNOS gene from the pBluescript Ⅱ SK-heNOS plasmid was performed,and an approximate 3.6 kbp objective fragment was achieved. The objective fragment was inserted into the downstream of the carrier promoter and combined with the report gene EGFP. The recombinant plasmid was verified by PCR of the bacterium liquid,restriction enzyme digestion and gene sequencing. Results:The heNOS fragment from pBluescript Ⅱ SK-heNOS plasmid were amplified and inserted into the pEGFP-N1 carrier. The enzyme digesting spectra of the recombinant plasmid was correct,and the heNOS gene sequence was completely in accordance with the sequence provided by the Genbank. Conclusion:The pEGFP-N1-heNOS eukaryotic expression recombinant plasmid were successfully constructed.