Stem cell-based therapies hold great promise in providing desirable solutions for diseases that cannot be effectively cured by conventional therapies. To maximize the therapeutic potentials, advanced cell tracking probes are essential to understand the fate of transplanted stem cells without impairing their properties. Herein, conjugated polymer (CP) nanodots are introduced as noninvasive fluorescent trackers with high brightness and low cytotoxicity for tracking of mesenchymal stem cells (MSCs) to reveal their in vivo behaviors. As compared to the most widely used commercial quantum dot tracker, CP nanodots show significantly better long-term tracking ability without compromising the features of MSCs in terms of proliferation, migration, differentiation, and secretome. Fluorescence imaging of tissue sections from full-thickness skin wound-bearing mice transplanted with CP nanodot-labeled MSCs suggests that paracrine signaling of the MSCs residing in the regenerated dermis is the predominant contribution to promote skin regeneration, accompanied with a small fraction of endothelial differentiation. The promising results indicate that CP nanodots could be used as next generation of fluorescent trackers to reveal the currently ambiguous mechanisms in stem cell therapies through a facile and effective approach.