The invention relates to the technical field of stem cell culture, in particular to a protein-free non-programmed freezing medium for umbilical cord mesenchymal stem cells as well as a preparation method and application of the protein-free non-programmed freezing medium. The cryopreservation solution comprises a basic solution, a nutritional supplement, a permeable protective agent, a non-permeable protective agent, a cell sedimentation stabilizer, a cell membrane protective agent, an apoptosis inhibitor and an antioxidant, the basic solution is DMEM / F-12 with the glucose content being smaller than or equal to 1000 mg / L. The cryopreservation liquid is suitable for directly cryopreserving umbilical cord mesenchymal stem cells at-80 DEG C after in-vitro amplification and before stem cell treatment. The cryopreservation liquid is free of serum and protein and clear in chemical component, and the cryopreserved umbilical cord mesenchymal stem cells are free of exogenous pollution risk and safer to use; after the cells are recovered, the viability is high, the adherence rate is high, and the cell expansion is fast; surface marker characteristics (phenotypes) and three-line differentiation potential of the mesenchymal stem cells can be maintained; optimization is carried out according to the cryopreservation and culture characteristics of the umbilical cord mesenchymal stem cells, the cryopreservation effect is improved, programmed cooling is not needed, and time and labor are saved.