Abstract： Objective To construct an eukaryotic vector with expression of human survivin gene with green fluorescent protein which is named pIRES2-EGFP/survivin and transfected into K562 cell line.Methods Using pDNR/Survivin plasmid as a template, the full length of survivin cDNA was amplified by PCR and subsequently cloned into T-A vector and then subcloned into pIRES2-EGFP vector. After identified by digestion of restrictive endonucleases, pIRES2-EGFP/survivin was further confirmed by sequencing. Then it was transfected into K562 cells with superfect reagents. The mRNA was isolated and survivin gene was detected by Western blotting. Results The exact sequences of pIRES2-EGFP/survivin vector were confirmed by digestion of restrictive endonucleases and sequencing. After transfection, the expressions of green fluorescent protein were present. The mRNA expression of survivin has been detected in transfected cells by RT-PCR. Conclusion The vector pIRES2-EGFP/survivin has been constructed and could express survivin gone in K562 cells successfully.