The invention discloses a subunit vaccine for preventing type-IV aviadenovirus as well as a preparation method and application thereof, which belongs to the field of veterinary biological products. The preparation method comprises the following steps: cloning and amplifying a C-end and an N-end structural domain genes of FadV 4C Hexon genes; establishing a recombinant baculovirus rBac-HEXON-P10-C-PH-N capable of co-expressing two antigen proteins by using an insect cell-baculovirus bidirectional expression system, wherein the recombinant virus efficiently expresses the FAdV 4C Hexon-C and Hexon-N antigen proteins in the insect cell HF; and extracting, purifying, BEI inactivation, and emulsifying by adding adjuvant, thus obtaining the vaccine. The preparation method is simple, a great amount of FAdV 4C Hexon-C and Hexon-N proteins can be simultaneously prepared, the consumed time is short, the expression amount is high, the production cost is greatly reduced, and the mass production canbe realized. The obtained recombinant subunit vaccine is good in immunity effect, small in immunity dose and capable of preventing the infection of the aviadenovirus.