Osteoarthritis is a common knee joint disease that significantly affects pet health, especially among domestic dogs. Bone marrow mesenchymal stem cells (BMSCs) are multipotent cells capable of differentiating into multiple lineages and self-renewing. However, their limited quantity and proliferative capacity during laboratory cultivation present challenges. In this study, BMSCs were isolated from the bone marrow of immature dogs and evaluated for various biological characteristics. Researchers assessed their ability to form colonies (Colony Forming Unit—CFU), stability of cellular morphology, proliferative capacity, expression of stem cell markers, and potential for differentiation into other cell types. The results indicated that the isolated cells exhibited colony-forming ability with a CFU-f of 5 ± 1 colonies per million mononuclear cells. The cells maintained stable morphology through the 8th passage and showed a doubling time of less than 42 hours. Additionally, these BMSCs expressed surface markers CD90 and CD105 while lacking CD34 and CD45, and they successfully differentiated into bone, cartilage, and fat cells. These findings encourage continued research into the clinical use of canine BMSCs for joint repair. In conclusion, this study presents a successful method for isolating and culturing canine BMSCs, highlighting their potential application in treating degenerative knee diseases.