Multifunctional superparamagnetic iron oxide nanoparticles are pivotal in bioapplications, with optimal size ranges varying by application. Exploring each size is essential to maximize functionality, as even 1-2 nm variations can significantly affect their properties. Therefore, discussing the effects of different sizes within the single-domain range of superparamagnetic ferrites is essential for understanding their performance in bioapplications. In this study, we synthesize monodisperse Fe3O4 nanoparticles with diameter ranging from 4.0 to 13.5 nm, the surface modified with PEGylated (Fe3O4-mPEG(2000); FP), and systematically evaluate size-dependent biobehavior and potential application of FP nanoparticles in SNU423 cells. The results reveal that specific loss power (SLP) is directly proportional to particle size, and the larger FP nanoparticles enable higher hyperthermal ablation efficacy in vitro, leading to more effective tumor growth inhibition in vivo. Meanwhile, particles with smaller sizes (< 8.5 nm) generate negligible heat, rendering them unsuitable for hyperthermal therapy, but optimal for magnetic resonance imaging (MRI). This work demonstrates that FPs nanoparticles with diameter of 13.5 nm exhibit a significant synergistic anticancer effect of magnetic hyperthermal therapy and effective T-2-weighted MRI with minimal side effects. This research presents important insights for nanoparticle design by precisely identifying the suitable size ranges for the biofunctions of Fe3O4 nanoparticles.