To establish a detection method of oncolytic adenovirus/p53 and standard of quality control, human telomerase reverse transcriptase (hTERT) promoter, CMV fusion promoter containing hypoxia reaction element (HRE) and p53 gene were identified by vector DNA restriction enzyme digestion and PCR analysis.  The result conformed that all modified regions were in consistent with theoretical ones.  Particle number was 2.0×10¹¹ mL⁻¹ determined by UV (A₂₆₀)_.  Infectious titer was 5.0×10¹⁰ IU·mL⁻¹ analyzed by TCID₅₀.  In vitro p53 gene expression in human lung cancer cell H1299 was determined by ELISA, and A₄₅₀ ratio of nucleoprotein in virus infection group to control group was 5.2.  Antitumor potency was evaluated by cytotoxicity assay using human lung cancer cell A549, and the MOI_IC50 of this gene therapy preparation was 1.0.  The tumor cells targeted replication ability of recombinant virus was determined by TCID₅₀ titer ratio of filial generation virus between human lung cancer cell A549 and human diploid epidermal fibrolast BJ cells after infected by virus with same MOI.  TCID₅₀ titer ratio of tumor cell infection group to normal cell infection control group was 398.  The IE-HPLC purity of virus was 99.5%.  There was less than 1 copy of wild type adenovirus within 1×10⁷ VP recombinant virus.  Other quality control items were complied with corresponding requirements in the guidance for human somatic cell therapy and gene therapy and Chinese pharmacopeia volume Ⅲ.  The detection method of oncolytic adenovirus/p53 was successfully established for quality control standard.  The study also provided reference for quality control of other oncolytic viral vector products.