Avian Influenza viruses (AIVs) are members of the family Orthomyxoviridae, genus influenza A virus. It causes enormous losses in the poultry industry worldwide. Infection with AIVs H5N1 and H9N2 were reported in many cases in poultry in Egypt. Effective vaccines can prevent infection. This work aimed to prepare and evaluate an inactivated bivalent AI (H5N1&H9N2) vaccine from the local Egyptian isolates using Montanide ISA 71 oil adjuvant. The prepared vaccines were sterile and safe. Cell mediated immune response of vaccinated chicks using lymphocyte blastogenesis and phagocytic activity showed high values from the 3rd day post vaccination (DPV) and continued till 21st DPV for monovalent (H5N1), monovalent (H9N2) and (bivalent H5N1&H9N2) AI vaccines. Serum antibody titer was increased from the 1st week post vaccination (WPV), and persisted in high values till 25th WPV against AIV H5N1 and AIV H9N2 for [monovalent H5N1 and bivalent H5N1&H9N2 AI vaccines] and [monovalent H9N2 and bivalent H5N1&H9N2 AI vaccines], respectively using Hemagglutination inhibition (HI) test. Vaccinated chicks showed reliable results upon challenge. In conclusion, the prepared AI vaccines on Montanide ISA-71 stimulate a good humoral and cellular immune responses, with the bivalent AI (H5N1&H9N2) could save cost for vaccine production and labor for vaccination as it stimulated superior early and high immune response compared with monovalent AI vaccines |