Abstract: The present invention discloses a serum-free medium for full suspension culture of MDCK cells and a preparation method of the serum-free medium. The serum-free medium for full suspension culture of the MDCK cells comprises basic metabolic nutrients, nucleotide, vitamins, inorganic salts, a shear force protective agent, a cell clustering resisting agent, a pH buffer agent, a pH indicator, an influenza virus proliferation accelerant and other additives. The preparation method of the serum-free medium for the full suspension culture of the MDCK cells comprises the following steps: 1) preparing a mixed solution: dissolving and mixing raw materials; and 2) regulating pH: regulating the pH of the mixed solution to 6.3 to 6.7, and setting a constant volume to obtain the serum-free medium for the full suspension culture of the MDCK cells.