As a novel duck adenovirus 3 (DAdV-3) infection caused significant economic losses to the poultry industry in China, there is an urgent need to develop a safe and effective vaccine. In the research, fiber-1 and fiber-2 proteins were expressed and purified, respectively. To evaluate the immunogenicity of the two recombinant proteins, we investigated the IgY antibodies and virus-neutralizing antibodies in duck sera. The protective efficacy was evaluated by mortality, virus shedding and histopathological examinations after challenged with the DAdV-3. Results showed that the IgY antibody levels of the fiber-2 group was significantly higher than that of the fiber-1 group and inactivated vaccine group. Ducks vaccinated with fiber-2 group provided full protection with no mortality, no virus shedding and no histological lesions, superior to other groups. These results suggest that the fiber-2 protein can be an ideal candidate for subunit vaccine against the disease.