Osteogenic differentiation is the basis of bone growth and repair related to many diseases, in which evaluating the degree and ability of osteogenic transformation is quite important and highly desirable. However, fixing or stopping the growth of cells is required for conventional methods to monitor osteogenic differentiation, which cannot realize the full investigation of the dynamic process. Herein, a new anion conjugated polymer featuring aggregation-induced emission (AIE) characteristics is developed with excellent solubility for monitoring the process of osteogenic differentiation. This novel polymer can bind with osteogenic differentiated cells, and the intracellular fluorescence increases gradually with the enhancement of osteogenic differentiation. Moreover, it possesses good biosafety with negligible effect on cell activity and osteogenic differentiation, which cannot be realized by the typical method of Alizarin Red S staining. Further study shows that the polymer crosses the cell membrane through endocytosis and enriches in lysosomes, whereas no obvious fluorescence is detected with other cells, including non-differentiated osteoblast cells, under the same conditions, demonstrating the high selectivity. This is the first fluorescent probe with excellent specificity to realize real-time observation of the process of osteogenic differentiation. Therefore, PTB-EDTA shows great promise in the study of osteogenic differentiation and related applications.