Abstract: OBJECTIVE To study on HEK293 cell to passage, establish 3-level cell banks, and by that to highly efficiency express a recombinant HPV16-E6E7 adenovirus vector. METHODS HEK293 cell passaged and established 3-level cell banks. And general inspected according to the requirements of Chinese Pharmacopeia Ⅲ 2010. By the HEK293 to propagate a recombinant HPV16-E6E7 adenovirus vector, identitied its titer, inserted target gene and genome macrorestriction. RESULTS HEK293 cell met the growthform of its description; the limited passages, concentration of cells, and install quantity of 3-level cell banks were all conformed to requirements of vaccine substrates; the expressed recombinant HPV16-E6E7 adenovirus titer was more than 3.0×10⁹ IU?mL^-1; the expressed inserted target gene and genome macrorestriction map were stable. CONCLUSION HEK293 cell and its 3-level cell banks established are all conformed to the requirements of vaccine substrates.