To construct an expression vector bearing hairpin ribozyme (Rz) gene against vascular endothelial growth factor (VEGF) and to assay the cleavage activity of the ribozyme in vitro. Methods: Anti-VEGF hairpin Rz gene was synthesized while VEGF gene was cloned from human placenta, and they were both inserted into the eukaryotic expression vector pcDNA3. Then pcDNA3-Rz and the pcDNA-VEGF were transcripted in vitro and cleavage activity of the Rz was measured. Results: The fragment of the Rz gene was confirmed by the digestion of pcDNA3-Rz with EcoR I and BamH I. The expression product of the Rz gene has higher cleavage activity. Conclusion:We have successfully constructed pcDNA3-Rz expressio n vector and laid a basis for further study of gene therapy.