The invention provides an I-group 4-type fowl adenovirus DNA vaccine and application thereof. According to the technical scheme, based on experimental means, research finds and shows that fiber protrusions have good immunity prototypes, in this way, with 4-type fowl adenovirus fibrous protein C-terminal genes being antigen substances, codon optimization is carried out on the basis of a natural sequence, an eukaryotic expression vector pCAGGS is cloned, and the DNA vaccine pCAGoptiFAV4C is constructed. According to the researched fowl adenovirus NDA vaccine, a method of gene engineering fermentation is adopted for preparing antigens, and therefore the I-group 4-type fowl adenovirus DNA vaccine is low in cost, pure in antigen and safe to use. By the utilization of a serology method and an immunity virus attack method, the immunity effect of the vaccine is evaluated. The result shows that the vaccine can provide effective immune protection for fowl, and has good commercial development prospects. Compared with a traditional vaccine, the DNA vaccine achieves the safety of subunit vaccines and inactivated vaccines, and also has the features of simultaneous induction of humoral immunity and cellular immune response, wherein the features only belong to attenuated vaccines or recombinant vaccines.