Objective To achieve the efficient isolation of mouse bone marrow mesenchymal stem cells (mBMSCs). Methods mBMSCs from C57BL/6 mice, aged 7 to 9 weeks, were isolated by four method: the bone fragment digestion and crawl method, whole-bone-marrow adherent method, bone fragment digestive fluid supernatant method, and bone fragment digestion and mortar method. An inverted microscope was used to observe the morphology of the mBMSCs, and their characteristic immunophenotype was detected by flow cytometry. The osteogenic and adipogenic differentiation abilities of mBMSCs were detected through multidirectional differentiation induction. Results Isolated primary cells were observed under an inverted microscope after the first fluid change. A large number of bone fragments and miscellaneous cells only were found in the primary cells isolated by the bone fragment digestion and crawl method, and this method was no longer evaluated. Only a small number of polygonal adherent cells and a large number of miscellaneous cells were observed in the whole-bone-marrow adherent-extracted sample. The cells isolated by the bone fragment digestive fluid supernatant method were longer spindle-shaped and triangular adherent cells, but there were many miscellaneous cells. The cells isolated by the bone fragment digestion and mortar method were longer spindle-shaped and polygonal adherent cells, and there were fewer miscellaneous cells. The isolated cells were cultured to passage 3, and the characteristic immunophenotypes of mBMSCs were analyzed by flow cytometry. The result showed that the purity of mBMSCs isolated via the bone fragment digestive fluid supernatant method and bone fragment digestion and mortar method was higher than that of cells isolated via the whole-bone-marrow adherent method. When osteogenic differentiation and adipogenic differentiation were induced, cells isolated by the bone fragment digestive fluid supernatant method and bone fragment digestion and mortar method had stronger multidirectional differentiation potential than those isolated with the whole-bone-marrow adherent method. Conclusions mBMSCs isolated using the bone fragment digestive fluid supernatant method and the bone fragments digestion and mortar method were of higher purity and quality than those from the other method.