Magnetic-fluorescent bifunctional drug delivery system which possesses magnetic targeting and fluorescent tracing capabilities, effectively improves the delivery efficiency of drugs. With the in-depth study of the properties of non-spherical microparticles, it is found that the shape of the microparticles also plays a key role in drug delivery. Because of the unique shape, rod-like microparticles have exhibited great drug molecule metabolic dynamics and excellent anti-tumor effects during the process of treatment. In this study, Fe₃O₄/NaYF₄: Eu³⁺/PLGA magnetic-fluorescent bifunctional microrods were prepared via one-step electrospraying. Unlike other methods that require complicated steps or expensive equipment, one-step electrospraying is a facile and low-energy method. The prepared magnetic-fluorescent bifunctional microrods possessed uniform rod-like morphology. Compared with magnetic-fluorescent bifunctional microspheres in same volume, it was found that the microrods showed a lower water contact angle. The results of hysteresis curve and fluorescence spectrum suggested the excellent magnetic and fluorescent properties of magnetic-fluorescent bifunctional microrods. After co-cultured with A549 cells or endothelial cells, the cell viability testing results confirmed the wonderful biocompatibility of microrods. When the drug doxorubicin was loaded by the immersion method, the microrods showed a higher drug entrapment efficiency and drug-loading capacity in comprasion with microspheres. In addition, microrods loaded with the same drug in weight showed stronger cytostatic effects after two days of co-culture with A549 cells. In summary, the magnetic-fluorescent bifunctional microrods prepared via one-step electrospraying will be promising candidates for biomedical applications in drug delivery, targeting and tracking.