The invention relates to a serum-free medium adapting to PK-15 all-suspended growth, a preparation method of serum-free medium and an all-suspended domestication method applied to a PK-15 cell. The serum-free medium comprises amino acid, Tween, linoleic acid, vitamin E, myristic acid, stearic acid, vitamin A, beta-mercaptoethanol, sodium chloride, glucose, cadmium chloride, linoleic acid, cholinechloride, inositol, ammonium metavanadate and the like. The provided serum-free medium can realize all-suspended growth of the PK-15 cell, so that the cost and digestion of a microcarrier are omitted,and the technical problem that the PK-15 cell microcarrier is difficult to digest and amplify in the suspended culture process is solved.