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Newcastle disease is a fatal viral disease which is highly contagious that affects most species of birds and is a major economic threat in the poultry industry. Both the HN and F glycoproteins of Newcastle disease virus (NDV) are essential for pathogenicity and virus infectivity. This study describes immunization of DNA vaccines encoding the HN, F or both the genes of New castle disease virus. In our previous study, the antigen expression of the insert genes has been validated in vitro by Western Blotting and Indirect Immunosenest. In this study, ELISA and HI analysis of the in vivo experiment on SPF (specific Pathogen Free) chickens showed the induction of humoral responses by the DNA vaccines. Our finding indicated that twice vaccination with pDNA was able to elicit significant antibody titers (P< 0.05) by either monocistronic (pIRES/HN and pIRES/F) or bicistronic (pIRES/F/HN) plasmid, after one week of second pDNA vaccination (booster). The results proposed that DNA immunization of chickens at second vaccination had enhanced the antibody response successfully. Also, it revealed that vaccination with the co-expression plasmid pIRES/HN/F can induce a stronger antibody response than vaccination with pIRES/HN or pIRES/F alone.