The invention discloses a construction scheme of artificially transforming human type 5 adenovirus (Ad5), and a specific application of a novel oncolytic adenovirus construct obtained by the scheme in tumor treatment, and belongs to the technical field of medical genetic engineering. A recombinant adenovirus construct obtained by PCR amplification site-directed deletion, enzyme digestion, ligation, cloning, homologous recombination, transfection, adenovirus monoclonal purification, etc., its technical characteristics are: Ad5 genome E1A conserved sequence 2 27 bases were deleted in the (CR2) region; 29477-29714nt of the ADP gene in the E3 region was deleted; and the full-length coding sequence (1131bp) of the HSV-TK gene was inserted in the deleted region. This construct is a new type of oncolytic adenoviral vector with higher tumor selective replication ability, which utilizes the suicide gene function of HSV-TK and the double killing effect of oncolytic virus to dissolve tumor cells, and has great potential in the biological treatment of tumors. Unique practical value.