Mesenchymal stromal cells (MSCs) hold significant promise in regenerative medicine, yet their clinical application is hindered by challenges such as cellular heterogeneity and quality control. This study aims to develop a rapid, noninvasive method for evaluating the quality of MSCs using femtosecond laser label-free imaging (FLI). We examined the proliferation, metabolic dynamics, and differentiation potential of MSCs from various tissue sources, including human dental pulp, umbilical cord, and fat, across different passages. Conventional experiments show that as the number of passages increases, the morphology of MSCs alters, proliferation capacity decreases, β-galactosidase activity linked to aging rises, and both osteogenic and adipogenic differentiation abilities markedly decline. FLI technology effectively captures these changes: reduced NAD(P)H/FAD ratio in higher-passage cells suggests decreased metabolic activity, while enhanced aging-related fluorescence signals, such as lipofuscin, align with cellular senescence. In the assessment of differentiation capability, increased fluorescence intensity of NAD(P)H and FAD signals indicates heightened metabolic activity within the cells. With the passages increasing, the fluorescence intensities of NAD(P)H and FAD decline, suggesting diminished ability of cell differentiation. Furthermore, during osteogenic differentiation, the optical REDOX ratio (FAD/(NAD(P)H + FAD)) decreases with successive passages, whereas during adipogenic differentiation, it increases. Three-dimensional FLI of suspension cells further reveals that the cells of lower-passage exhibit greater spatial heterogeneity in metabolic signals, possibly reflecting more active mitochondrial function. This study demonstrates that FLI technology can effectively assess the proliferation activity, senescence, and differentiation potential of MSCs through noninvasive, dynamic monitoring of their metabolic status and morphological features, offering a novel approach for standardized quality assessment of MSCs preparations.