The invention discloses an establishment method of a cLYZ/hTLF double-antibacterial gene recombinant adenovirus, and a recombinant adenovirus and application thereof. The method comprises the following steps: respectively amplifying cLYZ gene and hTLF gene from grouper head-kidney tissues and human milk somatic cell total RNAs, cloning the obtained goal genes into a pDC315 shuttle vector, co-transfecting HEK293 cells through liposome by using the target-gene-containing shuttle plasmid and adenovirus framework plasmid, and packaging to obtain the high-titer recombinant adenovirus; and evaluating the combined inhibiting actions of the cLYZ gene and hTLF gene on the milk cow mastitis primary pathogens under the adenovirus mediated conditions.