Abstract: The gB and gD gene of Infectious Laryngotracheitis virus (ILTV) Wang Gang (WG) strain were cloned into the eukaryotic expression vector pCAGGS and designated as pCAGG-gB, pCAGG-gD,respectively. Seventy-nine 30-day-old SPF white Leghorn chickens were randomly divided into seven groups ,five of them were immunized by four sites intramuscular injection with pCAGG-gB, pCAGG-gD, pCAGG-gB+pCAGG-gD，pCAGG-gB+rFPV-ILTVgB，rFPV-ILTVgB, respectively. Another two groups were injected with sodium chloride solution and pCAGGS as control groups. Two weeks after the second immunization, chickens were challenged intralaryngealy with 500EID50 of highly virulent ILTV WG strain. The sera antibodies detection result showed that all chickens immunized with pCAGG-gB, pCAGG-gD, pCAGG-gB+pCAGG-gD，pCAGG-gB+rFPV-ILTVgB，rFPV-ILTVgB developed ILTV-specific antibodies 2 weeks post-inoculation. The percentages of CD4+, CD8+ and TCRγδ+ T lymphocytes of these immunized groups were higher than control groups, and expression level of cytokine IFN-γ and IL-18 were higher in pCAGG-gB and pCAGG-gD groups than control groups. After challenge, chickens in control groups began to die at day 2 post-challenge. Chickens immunized with pCAGG-gB, pCAGG-gD had been partially protected (44%), the groups immunized with two plasmids (pCAGG-gB and pCAGG-gD) or plasmid and recombinant virus (pCAGG-gB+rFPV-ILTVgB) had higher protection (56%),the group immunized with recombinant virus (rFPV-ILTVgB) had the highest protection (69%). These results indicated that DNA immunization can induce immune response and partially protect chickens from homologous ILT virus challenge.