Egg-drop syndrome virus (EDSV) is an avian adenovirus that causes markedly decrease in egg production and in the quality of the eggs when it infects chickens. In this report, we engineered truncated fiber protein containing the entire knob domain and part of the shaft region as a vaccine candidate. The protein was obtained in the soluble fraction in Escherichia coli (E. coli), and expression level after nickel-affinity purification was 126 mg/L. By means of multiple characterization methods, it is demonstrated that the recombinant protein retains the native trimeric structure. A single inoculation with the structure-stabilized recombinant protein, even at the lowest dose of 2 μg, stimulated hemagglutination inhibition (HI) antibody responses in chickens, for at least 16 weeks. Neutralizing titers in sera from the protein immunized groups was similar to that of inactivated vaccine immunized group. The lymphocyte proliferation response and cytokine secretion were also induced in immunized SPF chickens. In addition, immunization with the fiber protein also significantly reduced the viral load in the liver. Taken together, these results suggest the truncated fiber protein as an effective single dose, long lasting and rapidly effective vaccine to protect against EDSV.