AIM: To investigate the designation, synthesis and biological activity of against vascular endothelial growth factor165 (VEGF165) ribozyme.METHODS: The ribozyme against VEGF165 was designed with computer. The transcriptional vector was constructed which included the anti-VEGF165 ribozyme and 5′, 3′ selfsplicing ribozymes. The hammerhead ribozyme and substrate VEGF~ mRNA were synthesized through transcription in vitro.The cleavage activity of the ribozyme on target RNA was observed in a cell-free system.RESULTS: The anti-VEGF165 ribozyme was released properly from the transcription of pGEMRz212 cleaved by 5′ and 3′self-splicing ribozymes which retained its catalytic activity,and the cleavage efficiency of ribozyme reached 90.7%.CONCLUSION: The anti-VEGF165 ribozyme designed with computer can cleave VEGF165 mRNA effectively.