Mesenchymal stem cell (MSC)-based therapy has emerged as a promising therapeutic strategy for tissue regeneration and repair. However, efficient targeted delivery to specific tissues remains an open challenge. Here, we non-invasively monitored the migration of MSCs labeled with Fe₃O₄@polydopamine nanoparticles (Fe₃O₄@PDA NPs) toward laser burn injury sites in a living rat model and evaluated the effects of the labeled MSCs at the injury site. The Fe₃O₄@PDA NPs could be effectively incorporated into the MSCs without any negative effects on stem cell properties. Furthermore, they enhanced the migration ability of the MSCs by up-regulating the expression level of C–X–C chemokine receptor type 4 (CXCR4). They also increased the secretion of some cytokines and the expression of healing-related genes in comparison with unlabeled MSCs. Labeled MSCs were intravenously administered into injured rats, and live imaging was performed to monitor MSC migration. Fluorescent signals of the labeled MSCs appeared at burn injury lesions 1 day after injection and then gradually increased up to 7 days. After 7 days, the group injected with the labeled MSCs showed less inflammation compared with those injected with the unlabeled MSCs. Additionally, the labeled MSC group showed increased cytokines and reduced pro-inflammatory factors compared with the unlabeled MSC group. The Fe₃O₄@PDA NPs enhanced stromal cell-derived factor-1/CXCR4-mediated MSC migration in vivo. Thus, we demonstrated the safety, feasibility, and potential efficacy of using the Fe₃O₄@PDA NPs for optimizing MSC-based therapeutic strategies for burn wound healing.