Objective To prepare Fe3O4 magnetic nanopaticles loaded poly(lactic-co-glycolic acid) (PLGA) microspheres using microfluidics, and to characterize this microspheres for future possible applications as targeted drug delivery and MRI tracking. Methods PLGA-Fe3O4 microspheres were prepared by three-phase microfluidic devices. Morphology of microspheres and distribution of Fe3O4 magnetic nanopaticles inside PLGA microspheres were analyzed. Results The PLGA-Fe3O4 microspheres prepared by microfluidic method had uniform particle size distribution and excellent dispersibility. The results of elemental analysis confirmed the presence of Fe, and the results of TEM observation showed that Fe3O4 magnetic nanopaticles were well dispersed inside the microspheres without agglomeration. Conclusion The feasibility of Fe3O4 magnetic nanopaticles loaded PLGA microspheres prepared by microfluidic method is demonstrated. The current study thus provides possibility of using PLGA microspheres for targeted drug delivery and MRI tracking.