The invention provides a cell culture medium, a preparation method and application, and belongs to the technical field of cell culture. The cell culture medium is prepared from a basic culture medium,cytokines, hormones, proteins, biogenic amines, an inhibitor and solution declining elements, wherein the cytokines, the hormones, the proteins, the biogenic amines, the inhibitor and the solution declining elements are added into the basic culture medium. The basic culture medium can effectively maintain the stability of the cell environment while maintaining the activity of the cells. By addingthe cytokines, the hormones, the proteins, the biogenic amines, the inhibitor and the solution declining elements into the basic culture medium, on the one hand, through the synergy between the components, animal-derived components in a traditional culture medium can be replaced, the stability of the cell culture medium can be ensured, and the risks of disease transmission and immune response arereduced; and on the other hand, the components cooperate with one another other, the cell activity is maintained, cells can proliferate steadily and rapidly, the primary separation efficiency of thecells is greatly improved, and the self-renewal and tissue repair capabilities of the cells are improved.