A recombinant adenovirus carrying the tumor suppressor gene PTEN (Ad5-PTEN) is an effective antitumor agent against liver cancer. But the application of Ad5-PTEN has been greatly hindered by its auto-immunogenicity, non-specific toxicity to normal tissues, as well as poor stability in blood stream because of neutralizing antibody. Epithelial cell adhesion molecule (EpCAM) is over-expressed in most solid tumors and it has been identified to be a cancer stem cell surface marker in liver cancer. An RNA aptamer EpDT3 could specially bind with EpCAM and target EpCAM-positive cells. Therefore, we hypothesized that developing a novel gene delivery system of EpDT3-modified Ad5-PTEN could overcome the disadvantages of naked Ad5-PTEN and enhance the antitumor effect on hepatocellular carcinoma. We took polyethylene glycol (PEG) as a linker to conjugate EpDT3 with Ad5-PTEN to prepare EpDT3-PEG-Ad5-PTEN (EPAP) by simple chemical synthesis method. We found that the stability of this novel gene delivery system in human blood serum increased about 16-fold compared to the naked adenovirus. Meanwhile, EPAP enhanced gene expression and cellular uptake in HepG2 cells, and showed significant inhibition in cell proliferation and cell migration against hepatocellular carcinoma cells HepG2 while showing no cytotoxicity to normal liver cells L-02, compared with Ad5-PTEN. Importantly, EPAP could induce cell apoptosis and presented superior antitumor activity against aggressive HepG2 xenograft in nude mice but showed no obvious toxicity to the tested mice at the therapy concentration. In conclusion, EpCAM aptamer EpDT3 could significantly enhance the antitumor effect of Ad5-PTEN with high binding ability to EpCAM-positive cells HepG2.