Objective To establish an efficient method for isolation and culture of rat bone marrow mesenchymal stem cells (BMSCs), and apply PKH26 to label them in vitro to explore the effect of PKH26 labeling on the biological characteristics of BMSCs, as well as the in vitro tracing. Methods The bone of both hind limbs of 5d suckling rats were separated, the surrounding muscle and fascia were removed, and cut into small pieces for culture. BMSCs were purified by fluid exchange and passage, and the third generation cell surface antigen was determined by flow cytometry. Under the same culture conditions, the third generation BMSCs were labeled with PKH26. Cell morphology and proliferation status were observed under fluorescence microscope in the labeled group and the unlabeled group, and the adipogenic induction characteristics and identification of the labeled group and the unlabeled group were compared. Results The bone marrow slice method was used to separate the hind limb bones of suckling mice. The BMSCs were slender spindle shaped and uniform in shape. A large number of BMSCs could be rapidly obtained in a short time; The results of flow cytometry showed that the expression of CD29 was (91.18 ± 1.29)%, the expression of CD90 was (91.18 ± 1.29)%, and the expression of CD45 was (1.74 ± 0.36)%; PHK26 labeling had no significant effect on the morphology and proliferation of BMSCs cells (P>0.05), and had no effect on the induction of osteogenesis and adipogenesis. Conclusions A large number of high-purity BMSCs can be rapidly cultured by the method of 5-day rat bone marrow slices, which can be used as seed cells for bone tissue engineering; PKH26 can label rat BMSCs in vitro.