Magnetic iron oxide nanoparticles (MIONs) have unique physical-chemical properties for a wide range of biomedical applications, including targeted drug delivery, multimodality molecular imaging, thermal therapies, and biomarker detection. In this study, I optimized MIONs for different biomedical applications. First, to improve the efficacy of cancer immunotherapy for solid tumors, I increased endocytosis of MIONs into T cells using cell penetrating peptides to enable better in vivo imaging and targeting. Secondly, to facilitate hyperthermia therapy and anti-cancer drug delivery by heat-triggered release, I coated MIONs with mesoporous silica and demonstrated enhanced colloidal stability and heating efficiency. Thirdly, to measure the levels of trace biomarkers for early cancer detection with high sensitivity, I quantified pancreatic cancer biomarkers in patient serum samples using europium-doped iron oxide nanoparticle-linked immunosorbent assay (ILISA) and optimized the detection limit of the assay. Our work further expanded the translational potentials of MIONs.