Newcastle and Influenza diseases are important viral diseases and its occurrence and virulence in Iran has increased in recent years. The purpose of this study was to evaluate the humoral immune responses of chickens vaccinated with inactivated dual oil emulsion Newcastle disease and avian influenza vaccine in two methods of intramuscular and subcutaneous injection as well as to assess the possible changes in serum biochemical factors. In this study, after subcutaneous and intramuscular inoculation of inactivated dual oil emulsion Newcastle-Influenza vaccine, the serum antibody level and hematobiochemical factors of these avian were analyzed on 7, 14, 21, and 28 days after vaccine injection.The results showed that the values of biochemical parameters such as albumin, glucose, total protein, triglyceride, cholesterol, ALT, AST, and ALP enzymes, sodium and potassium minerals, and hematocrit and hemoglobin levels in vaccinated and non-vaccinated chickens were not significantly different. Also, in chickens injected with antigen, Newcastle disease and influenza antibody titers were significantly different with those groups containing adjuvant.The results of this study indicate that the intramuscular and subcutaneous injection of dual combination vaccine is similar in terms of changes in biochemical and hematological factors and Newcastle disease and influenza antibody titers. To ensure safety in inactivated vaccines, the presence of proper adjuvant is essential for immune response.