Avian influenza (AI) was caused by A avian influenza virus in chicken all over the world. One 1.6 kb DNA fragment was amplified by RT-PCR from the RNA of AIV A/Chicken/Henan/1/1999(H9N2) and proved to be HA gene by sequencing. The HA gene expression plasmid pVAX-H9 was constructed by inserting the fragment into the pVAX1 vector. 3-week-old SPF chickens were inoculated with 50 μg DNA of plasmid pVAX-H9 by electroporation, 5 weeks later, all chickens were challenged with 100×EID50 of AIV A/Chicken/Henan/1/1999(H9N2), 1 week post challenge all birds were sampled by cloacal swabbing to isolate virus. The virus isolation was negative in all vaccinated birds and positive in all control birds. The HI titers reached to 10lg2 in vaccinated group, in contrast to 2lg2～4lg2 in control group post challenge respectively. These results showed that the plasmid pVAX-H9 designed as DNA vaccine would be able to elicit a firm protective immune response against AIV infection.