The invention discloses a serum-free medium for full-suspension culture of MDCK cells (madin-darby canine kidney cells) and a preparation method of the serum-free medium. The serum-free medium for full-suspension culture of the MDCK cells consists of a basic metabolism nutrient, nucleotide, vitamins, inorganic salt, 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; and the preparation method of the serum-free medium for full-suspension culture of the MDCK cells comprises the following steps: 1) preparing mixed liquid: dissolving and mixing the raw materials; and 2) regulating pH: regulating pH of the mixed liquid to 6.3-6.7, and setting a constant volume, so that the serum-free medium for full-suspension culture of the MDCK cells is obtained. The medium supports the high-density full-suspension culture of the MDCK single cells and is capable of greatly shortening a time of domesticating the MDCK cells from adherent cells into the serum-free full-suspension cells; and the medium is applicable to the large-scale production of biological products, in particular veterinary biological products.