The low pathogenic avian influenza viruses (LPAIV) subtype H9N2 are highly prevalent in the Middle East and are causing economic losses in the commercial poultry production sectors. Due to potential of H9N2 infections in human, LPAI H9N2 poses serious public health risk. Therefore, development of new potent and cost-effective influenza vaccines is urgently needed to safeguard poultry and to contain zoonosis. The primary goal of this study was to develop H9 plasmid-based DNA vaccine against the currently circulating AIV subtype H9N2 targeting HA gene of the virus A/Turkey/Egypt/1341V/2013(H9N2). The full length sequence of HA gene was codon optimized to the chicken biased codons and subcloned into the pCAGGS plasmid vector under the control of the chicken β-actin promoter. The in vitro expression of recombinant plasmid DNA was performed by transfection of 293T (HEK) cell line. The HA protein was analyzed using SDS-PAGE followed by Western blot and immunofluorescence assays. The pCAGG-optiH9 vaccine efficacy was evaluated by intramuscular immunization of SPF chickens with different concentrations of plasmid DNA and the sera were collected weekly post vaccination for antibody detection by HI test. All immunized chickens shown high HI antibody titers (9Log2) two weeks post-booster dose. The chickens were then challenged using homogenous AI H9N2 strain. During the course of infections, oropharyngeal and cloacal swabs were collected from all chickens at 3, 5 and 7 days post-challenge (p.c.) for virus titration in eggs. Data indicated that all chickens vaccinated with pCA-Egy-H9 were fully protected without clinical signs and virus shedding. Our results revealed that the pCA-Egy-H9 plasmid DNA vaccine could induce complete protection in chickens against H9N2 virus challenge and may propose an alternative strategy to control virus infections in the poultry industry.