Aim To clone human vascular endothelial growth factor-165 (VEGF-165) gene and human fibroblast growth factor-2 (FGF-2), and clone core hypoxia-response enhancer RTP801 (RTP801HRE) of RTP801 promoter. Using plasmid pIRES as backbone to construct eukaryotic expression vector, then transfecting it into human embryo kidney 293 cell and observing the expression of VEGF-165 and FGF-2 under normal condition and anoxic condition. Methods The 337 bp～511 bp of core hypoxia-response enhancer (core HRE) in RTP801 promoter was obtained by polymerase chain reaction (PCR) from mouse genomic DNA, to replace the CMV enhancer (150 bp～390 bp) in pIRES. The fragment containing VEGF-165 and FGF-2 was acquired by PCR, and then inserted into the recombinant plasmid pIRES/Igκ-VEGF-165/IRES/Igκ-FGF-2, and transfected recombinant plasmid pIRES/RTP801HRE/Igκ-VEGF165/IRES/Igκ-FGF-2 into the 293 cells with biodegradable hyperbranched polyethylenimine (PEI) in vitro. The infected cells were cultivated for 36 hours under normal and anoxic condition respectively. The expression of VEGF-165 and FGF-2 were detected by Western blot and ELISA. Results Eukaryotic expression vector pIRES/RTP801HRE/Igκ-VEGF-165/IRES/Igκ-FGF-2 has been confirmed to be successfully constructed by PCR, enzyme digestion and sequencing. Highly efficient co-expression of VEGF-165 and FGF-2 by the recombinant plasmid under the regulation of the RTP801 core enhancer in anoxic 293 cells has been shown by both Westernblot and ELISA. Conclusion The RTP801 core hypoxia-response enhancer can enhance the expression of downstream genes, so the constructed eukaryotic vector which contains this enhancer can high-efficiently express VEGF-165 and FGF-2 under anoxic condition