In the past decades, biomaterials were designed to induce stem cell toward osteogenic differentiation. However, conventional methods for evaluation osteogenic differentiation all required a process of cell fixation or lysis, which induce waste of a large number of cells. In this study, a fluorescence nanoprobe was synthesized by combining phosphorylated fluoresceinamine isomer I (FLA) on the surface of mesoporous silica-coated superparamagnetic iron oxide (Fe3O4@mSiO2) nanoparticles. In the presence of alkaline phosphatase (ALP), the phosphorylated FLA on the nanoprobe would be hydrolyzed, resulting in a fluorescence recovery of FLA. During early osteogenic differentiation, a high-level expression of cellular ALP was induced, which accelerated the hydrolysis of phosphorylated FLA, resulting in an enhancement of cellular fluorescence intensity. This fluorescence nanoprobe provides us a rapid and non-toxic method for the detection of cellular ALP activity and early osteogenic differentiation.