Abstract: In order to study the synergistic effect of chicken interleukin-4（IL-4） and IL-2 recombinant gene adjuvant pCI-chIL-4-chIL-2-EGFP on chicken coccidiosis live vaccine, the immune challenge test was carried out by combining genetic adjuvant with chicken coccidiosis live vaccine. The minimum effective dose and route of administration of the gene adjuvant were determined, and the effects of the gene adjuvant on the shortest immune interval time and immune generation period of chicken coccidiosis live vaccine were determined. The results showed that anticoccidial index（ACI） and relative weight gain rate（RWG） increased with the increase of the dose of gene adjuvant, and the ACI（189.32） and RWG（90.35%） reached the peak when the dose of gene adjuvant was at 100 μg/chinken. The ACI（188.41） of the oral 300 μg/gene adjuvant group was close to that of the intramuscular 100 μg/gene adjuvant group. The shortest time between two immunizations（6 d） in gene adjuvant challenge group（100 μg/chinken） was shorter by 1 d than that in gene adjuvant challenge group（7 d）. The immune generation period of the intramuscular adjuvant immune challenge group was 5 days after the second immunization, 1 d earlier than that of the non-gene adjuvant immune challenge group. These results indicated that intramuscular injection of 100 μg/chinken with pCI-chil-4-Chil-2-EGFP, IL-4 and IL-2 recombinant gene adjuvant could improve the immune effect of chicken coccidiosis live vaccine and shorten the immune generation period and interval.