The invention discloses a recombinant adenovirus and tetravalent adenovirus vaccine and a preparation method thereof. The tetravalent recombinant adenovirus vaccine contains a recombinant type 3 adenovirus strain, a recombinant type 7 adenovirus strain, a recombinant type 14 adenovirus strain and a recombinant type 55 adenovirus strain. The preparation method disclosed by the invention comprises the following steps: preparing recombinant shuttle plasmids containing hexon gene segments, and performing in-bacteria homologous recombination with a recombinant human type 3 adenovirus strain, thereby obtaining a recombinant adenoviral genome in which the hexon gene segments are replaced by type 7, type 14 and type 55; transfecting cells, rescuing to obtain recombinant human type 3, 7, 14 and 55 recombinant adenoviruses with different main capsid protein-hexon proteins; purifying, mixing according to the same protein content, and inactivating by using beta-propiolactone, thereby obtaining the tetravalent adenovirus vaccine. The tetravalent adenovirus vaccine is capable of inducing neutralizing antibody responses to four types of serotype adenoviruses, and the neutralizing titer is 500-1000.