Abstract: Objective To construct pAAV-TK-IRES-ES and identify its function. Methods①IRES,TK,ES framents from pIRES-MCS, pAAV-TK, pAAV-ES were attained by PCR and then cloned into vector pMD-19T simple to construct pAAV-TK-IRES-ES.②Viral particle of purified rAAV were assayed by AVSachTM ELISA.③Identify the primary function of r AAV-TK-IRES-ES via T24 cell and HUVEC cell Result ①pAAV-TK-IRES-ES was constructed and tested correctly by sequence indentification and enzyme digestion.②We obtained high quality of rAAV after dissociating and purifying. The viral particles title of rAAV were 2×1011-12 v.p/ml. ③ rAAV-TK-IRES-ES has both functions of endostatin and suicide gene by inducing cell apoptosis of T24 and HUVEC cell. Conclusion We successfully constructed rAAV-TK-IRES-ES and it can inhibit tumor induced angiogenesis and suppress both the initiation and the subsequent growth of human bladder cancer.