Multifunctional magnetic nanoparticles and derivative nanocomposites have aroused great concern for multimode imaging and cancer synergistic therapies in recent years. Among the rest, functional magnetic iron oxide nanoparticles (Fe 3O 4 NPs) have shown great potential as an advanced platform because of their inherent magnetic resonance imaging (MRI), biocatalytic activity (nanozyme), magnetic hyperthermia treatment (MHT), photo-responsive therapy and drug delivery for chemotherapy and gene therapy. Magnetic Fe 3O 4 NPs can be synthesized through several methods and easily surface modified with biocompatible materials or active targeting moieties. The MRI capacity could be appropriately modulated to induce response between T 1 and T 2 modes by controlling the size distribution of Fe 3O 4 NPs. Besides, small-size nanoparticles are also desired due to the enhanced permeation and retention (EPR) effect, thus the imaging and therapeutic efficiency of Fe 3O 4 NP-based platforms can be further improved. Here, we firstly retrospect the typical synthesis and surface modification methods of magnetic Fe 3O 4 NPs. Then, the latest biomedical application including responsive MRI, multimodal imaging, nanozyme, MHT, photo-responsive therapy and drug delivery, the mechanism of corresponding treatments and cooperation therapeutics of multifunctional Fe 3O 4 NPs are also be explained. Finally, we also outline a brief discussion and perspective on the possibility of further clinical translations of these multifunctional nanomaterials. This review would provide a comprehensive reference for readers to understand the multifunctional Fe 3O 4 NPs in cancer diagnosis and treatment.