The invention provides a preparation method and application of a passage cell origin newcastle disease (ND), infectious bronchitis (IB) and avian influenza (AI) triple inactivated vaccine. The methodincludes the steps of: subjecting an EB66 cell to growth culture in a bioreactor for virus inoculation; employing a two-stage culture method to inoculate newcastle disease virus, infectious bronchitisvirus or avian influenza virus to the EB66 cell; after 24h of virus inoculation, harvesting and storing the virus when the virus EID50 reaches a highest level, thus obtaining a virus liquid; repeating the steps to obtain the newcastle disease virus liquid, infectious bronchitis virus liquid and avian influenza virus liquid respectively, and conducting inactivation; mixing the inactivated newcastle disease virus liquid, infectious bronchitis virus liquid and avian influenza virus liquid to prepare the inactivated vaccine. The method provided by the invention employs the EB66 passage cell linefor culture of the newcastle disease virus, infectious bronchitis virus and avian influenza virus, avoids the risk of embryonic heterogenous protein and exogenous virus pollution caused by use of chicken embryos, improves the virus production quality, and simplifies the virus production process.