Egg-drop syndrome (EDS) virus is an avian adenovirus that causes a sudden drop in egg production and in the quality of the eggs when it infects chickens， leading to substantial economic losses in the poultry industry. Inactivated EDS vaccines produced in embryonated duck eggs or cell culture systems are available for the prophylaxis of EDS. However， recombinant subunit vaccines that are efficacious and inexpensive are a desirable alternative. In this study， we engineered chimeric fusion proteins in which the trimeric fiber knob domain lacking the triple β-spiral motif in the fiber shaft region was genetically fused to trimeric coiled coils， such as those of the engineered form of the GCN4 leucine zipper peptide or chicken cartilage matrix protein (CMP). The fusion proteins were expressed predominantly as soluble trimeric proteins in Escherichia coli at levels of 15-80mg/L of bacterial culture. The single immunization of chickens with the purified fusion proteins， at a dose equivalent to 10μg of the knob moiety， elicited serum antibodies with high hemagglutination inhibition (HI) activities， similar to those induced by an inactivated EDS vaccine. A dose-response analysis indicated that a single immunization with as little as 1μg of the knob moiety of the CMP-knob fusion protein was as effective as the inactivated vaccine in inducing antibodies with HI activity. The immunization of laying hens had no apparent adverse effects on egg production and effectively prevented clinical symptoms of EDS when the chickens were challenged with pathogenic EDS virus. This study demonstrates that the knob domain lacking the shaft sequence but fused to a trimeric coiled coil is a promising candidate subunit vaccine for the prophylaxis of EDS in chickens.