Abstract The main goals of the present investigation are to prepare a viral DNA vaccine to help stimulate the immune system of poultry and to increase the efficiency of this vaccine. To accomplish this work, a strain of H5N1 circulating in Egypt was confirmed using rapid diagnostic methods and also using reverse transcriptase polymerase chain reaction (RT-PCR) to hemagglutinin and neuraminidase genes. The virus was propagated in MDCK cell line and the viral genes were extracted, reverse transcribed and individually. Individual genes was cloned in gene expression vector (PHW2000) and used as DNA vaccine. The level of maternal antibodies was determined by ELISA to appoint the right time to give the vaccine. The chicks were divided into eight groups, each group was vaccinated by couple of DNA NP with one of another genes. The efficiency of coupled DNA vaccine was determined by neutralization assay and compared with inactivated vaccine. The results showed that the vaccine that consists of NP with NS has adequate protection for poultry.