This study aimed to comprehensively evaluate the biological activity in different passage populations of mesenchymal stem cells (BMSCs) derived from bone marrow in ovariectomy osteoporotic rats (named OVX-rBMSCs), providing experimental basis for new osteoporotic drug development and research. OVX-rBMSCs were isolated and cultured in vitro by the whole bone marrow adherent screening method. The morphological observation, cell surface markers (CD29, CD45, CD90) detection, cell proliferation, induced differentiation experimental detection were performed to evaluate the biological activity of Passage 1, 2, 3, 4 populations (P1, P2, P3, P4) OVX-rBMSCs. The results showed that whole bone marrow adherent culture method isolated and differentially subcultured OVX-The morphology of P4 OVX-rBMSCs was identical fibroblast-like and had the characteristics of ultrastructure of stem cells. The CD29 positive cells rate, CD90 positive cells rate, cell proliferation index, and the osteogenic, adipogenic, chondrogenic differentiation capacities of P4 OVX-rBMSCs were significantly better than those of other populations (P < 0.05). OVX-rBMSCs purity and biological activity were gradually optimized with the passaged, and among them P4 cells were superior to all the other populations. Based on these results, we report that the P4 OVX-rBMSCs model developed in this study can be used to develop a new and effective medical method for osteoporotic drug screening.