Fe3O4 magnetic nanoparticles possess good biocompatibility,large specific surface area, low toxicity,and high saturation magnetization,etc,so they are widely used in biomedical applications. The preparation methods of Fe3O4,including co-precipitation method,microemulsion method,high temperature heat decomposition method,hydrothermal method,sol-gel method and biosynthesis,are introduced in this paper and the advantages and disadvantages of co-precipitation method,hydrothermal method and high temperature heat decomposition method are also compared. The functionalization of Fe3O4 magnetic nanoparticles are reviewed, including small organic molecules,organic polymers,and inorganic nanomaterials. The application and research of Fe3O4 magnetic nanoparticles in biomedical fields are reviewed,including magnetic resonance imaging, drug delivery systems,protein and nucleic acid isolation,blood purification and tumor hyperthermia. Finally,some problems and perspectives in these research areas are outlined.