Abstract: Tattooing is one of the economical and effective DNA delivery methods，which is an invasive procedure involving a solid vibrating needle that repeatedly punctures the skin, wounding both the epidermis and the upper dermis in the process and causing cutaneous inflammation followed by healing, and thus nonspecifically stimulates the immune system. In this study, we focused on revealing the difference of two routes of administrations of DNA vaccine (intradermal tattoo versus intramuscular injection) on induction of immune response on BALB/c mice and SPF chickens, aiming at providing a new idea for DNA immunization. For comparison,we used eukaryotic expression vectors pCDNA3.1gJ (containing gJ gene of infectious laryngotracheitis virus) and pVAX1F (containing F gene of Newcastle disease virus) as the model antigen. BALB/c mice were inoculated with recombinant plasmid pCDNA3.1gJ and SPF chickens were inoculated with recombinant plasmid pVAX1F via intradermally by tattooing and intramuscularly by needle injection. Serum samples were collected regularly postvaccination for detection of the specific IgG antibody level. The chickens were challenged by NDV F48E9 strain with a dose of 105EID50 two weeks after the third immunization, then their morbidity and mortality were recorded. The results showed that the humoral immune responses elicited by tattooing are significantly stronger than elicited by intramuscular injection both on BALB/c mice and on chickens. In addition, low dose(50 μg )DNA via tattooing can induce higher specific antibody level than high dose(100 μg) via intramuscular injection in the same period,and tattooing immunizations can provide better immune protection than intramuscular injection. In summary, the tattoo delivery of DNA vaccine is a costeffective method and can induce more rapid and more robust immune responses that may meet the laboratory conditions need,which also provides a new idea for the future research directions on DNA immunization.