Hollow iron oxide nanoparticles (NPs) are an attractive class of hollow nanostructures that have received significant attention in the biomedical field due to their low toxicity, good biocompatibility, and intrinsic magnetic nature. We review the recent advances in the preparation, surface functionalization, and biomedical applications of hollow iron oxide NPs. Hollow iron oxide NPs are generally synthesized by the following five strategies, including the Kirkendall effect, galvanic replacement, chemical etching, nano template-mediated, and hydrothermal/solvothermal routes. We also summarize the general strategies for iron oxide NP surface functionalization. Moreover, various promising biomedical applications of hollow iron oxide NPs, including magnetic resonance imaging, drug delivery, and cancer therapy, are highlighted in detail. Finally, perspectives of hollow iron oxide NPs are provided.