The invention relates to canine umbilical cord mesenchymal stem cells as well as a preparation method and a cryopreservation method thereof. Specifically, the method comprises the following steps: disinfection and washing, digestion treatment, cell culture, cell passage, and cell cryopreservation. The method for preparing mesenchymal stem cells from a canine umbilical cord exhibits an excellent technical effect. The mesenchymal stem cells is beneficial for treatment of canine arthritis, fractures, muscle damage, ligament injury, cartilage damage, joint damage, cognitive dysfunction, immune-mediated diseases, dry eye, recurrence uveitis, liver disease, heart disease, kidney disease, diabetes, gastrointestinal disease, thyroid disease and skin disease.