The invention belongs to the technical field of veterinary biological products and particularly relates to a preparation method of avian influenza virus subtype H9 and fowl adenovirus 4 combined inactivated vaccine. LHM (Leghorn male hepatocarcinoma) continuous cell line is used as carrier cells to perform viral multiplication, culture medium A composed of glutamine, recombinant human insulin, human serum albumin, transferrin, biotin and growth factors is used to perform culturing, and a viral liquid is collected and inactivated to prepare the vaccine. The multiplication of avian influenza virus subtype H9 and fowl adenovirus 4 by the LHM continuous cell line has no need for additional pancreatin, the background of LHM cells is clear, no extraneous pathogens occur, the multiplication is easy, the process can be effectively simplified, and the cost can be reduced; in addition, the avian influenza virus and fowl adenovirus combined inactivated vaccine has high viral content, good stability and high safety and is an ideal avian influenza virus and fowl adenovirus combined inactivated vaccine.