In an attempt to investigate the immune efficacy of a DNA prime–protein booster strategy against avian coccidiosis with a chimeric construct, the Eimeria tenella antigen gene (3-1E) and chicken interferon gamma gene (ChIFN-γ) were subcloned into the mammalian expression vector proVAX forming the plasmids proE and proI, and then linked by splicing overlap extension by polymerase chain reaction to construct the chimeric plasmid proIE; the chimeric protein (rIE) was expressed in Escherichia coli harboring the constructed plasmid pGEX/IE. Broilers were administered two intramuscular injections with the constructed DNA vaccines (50 μg); in the protein booster groups 100 μg of the rIE were given following the proIE prime. After challenge the proIE-vaccinated chickens showed the protective immunity as demonstrated by significantly reduced oocyst shedding compared with chickens immunized with proE, but the proIE vaccine did not have an additive effect of increasing antibody titer and body weight gain. The chickens in the rIE booster groups had significantly higher specific antibody responses than those immunized with proIE, and displayed further decreased oocyst shedding and increased body weight gain. Taken together, these results indicate that ChIFN-γ exerts an adjuvant effect coexpressed with 3-1E and provide the first evidence that the DNA prime–protein booster strategy is able to augment the protective efficacy of chimeric DNA vaccine against challenge with Eimeria tenella.