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Liver cancer is a fatal disease with limited therapy options. The recombinant adenovirus expressing tumor-suppressor gene of PTEN (Ad5-PTEN) showed effective antitumor activity against liver cancer. However, its disadvantages produced great limitation on its application, especially its nonspecific and toxicity to normal cells and tissues. The epithelial cell adhesion molecule (EpCAM) is over-expressed in some liver cancer cells and an RNA aptamer EpDT3 could specially target to EpCAM-positive cells. Based on this founding, we aimed to design a kind of gene delivery system of EpDT3-mediated Ad5-PTEN (EpDT3-PEG-Ad5-PTEN, EPAP) in which polyethylene glycol was used to be a linker to conjugate EpDT3 with Ad5-PTEN. This strategy may overcome the disadvantages of naked Ad5-PTEN and enhance the antitumor effect on liver cancer. The SDS-PAGE electrophoresis, TBE-PAGE electrophoresis and fluorescence detection were conducted to confirm the successful preparation of EPAP. Compared with the naked Ad5-PTEN, EPAP showed significant anti-proliferative and anti-migratory activities against HepG2 cells. EPAP also showed selective and precise target ability to EpCAM-positive HepG2 cells in vivo. Therefore, EPAP may be further explored as a novel effective anticancer drug for malignant liver cancer.