The invention provides dog mesenchymal stem cell injection and a preparation method and application thereof. The dog mesenchymal stem cell injection comprises dog mesenchymal stem cells, heparin calcium, dog blood albumin and an electrolyte solution. The mesenchymal stem cells from dog bone marrow are utilized ingeniously, and combined with other compositions to be prepared into an injection form, namely the dog mesenchymal stem cell injection. The traditional therapeutic method for a dog skin tissue injury is broken. A stem cell therapy is brought in the field of pet medical treatment, the dog mesenchymal stem cell injection has the advantages that stability is high, the curative effect is good, usage is safe, long-term preservation and transportation are achieved, no toxic and side effect exists, and the foundation is laid for large-scale clinic use.