The invention discloses a human umbilical cord mesenchymal stem cell serum-free culture medium and a preparation method and application thereof. The human umbilical cord mesenchymal stem cell serum-free culture medium comprises a basic culture medium, amino acid substances, trace elements, recombinant human serum albumin, transferrin, recombinant human insulin, recombinant human epidermal growth factors, alkaline fibroblast growth factors and buffer salt, wherein the amino acid substances comprise glycine, alanine, asparagine, aspartic acid, glutamic acid, proline, serine and glutamine. The serum-free culture medium is clear in components, stable in batch, free of serum, antibiotics and other serum analogues, and free of heterogeneous pollution and immunological rejection risks and safe touse; by the serum-free culture medium, normal cell adherence and good cell growth state can be kept; the serum-free culture medium has stemness and is suitable for the growth and stemness maintenanceof human umbilical cord mesenchymal stem cells.