BACKGROUND Oncolytic viruses (OVs) can specifically infect and kill tumor cells. Adeno-associated virus (AAV) is a widely-studied OV. This study aimed to construct a tumor-targeted recombinant AAV using genetic engineering technology. MATERIAL AND METHODS The transgene plasmid pAAV-HE1B19K-TE1A was constructed with 4 genes (hTERT, E1A, HKII, and E1B19K) and co-transfected with pAAV-RC and pHelper to tumor cells (HepG2, A549, BGC-803) and normal cells (HUVEC). rAAV was verified with fluorescence microscopy. Quantitative PCR (qPCR) assay was used to test the titer of rAAV in each cell line. Apoptosis was analyzed using qPCR and Western blot assay. MTT was used to detect the effect of rAAV on cell viability. RESULTS The pAAV-HE1B19K-TE1A transgene plasmid was successfully structured. pAAV-HE1B19K-TE1A was highly expressed in all tumor cells. The titers of pAAV-HE1B19K-TE1A in HepG2, A549, and BGC-803 were 7.4×10⁷, 1.4×10⁸, and 1.1×10⁸ gc/μl, respectively. pAAV-HE1B19K-TE1A significantly decreased cell viability of tumor cells compared to that in HUVEC (p<0.05). pAAV-HE1B19K-TE1A remarkably triggered cleaved caspase 3 (C-caspase 3) activity in tumor cells compared to that in untransfected tumor cells (p<0.05). pAAV-HE1B19K-TE1A significantly induced release of cytochrome C (Cyto C) in tumor cells compared to that in untransfected tumor cells (p<0.05). pAAV-HE1B19K-TE1A demonstrated no toxicity to vital tissues of animals. CONCLUSIONS Tumor-targeted rAAV was successfully produced using the Helper-free system with recombinant plasmid, demonstrating high efficacy in decreasing viability of tumor cells without adverse effects on normal cells.