Despite its wide establishment over the years, iron oxide nanoparticle (IONP) still draws extensive interest in the biomedical fields due to its biocompatibility, biodegradability, magnetivity and surface tunable properties. IONP has been used for the MRI, magnetic targeting, drug delivery and hyperthermia of various diseases. However, their poor stability, low diagnostic sensitivity and low disease-specificity have resulted in unsatisfying diagnostic and therapeutic outputs. The surface functionalization of IONP with biocompatible and colloidally stable components appears to be promising to improve its circulation and colloidal stability. Importantly, through surface functionalization with designated functional components, IONP-based assemblies with multiple stimuli-responsivity could be formed to achieve an accurate and efficient delivery of IONP to disease sites for an improved disease diagnosis and therapy. In this work, we first described the design of biocompatible and stable IONP assemblies. Further, their stimuli-driven manipulation strategies are reviewed. Next, the utilization of IONP assemblies for disease diagnosis, therapy and imaging-guided therapy are discussed. Then, the potential toxicity of IONPs and their clinical usages are described. Finally, the intrinsic challenges and future outlooks of IONP assemblies are commented. This review provides recent insights into IONP assemblies, which could inspire researchers on the future development of multi-responsive and disease-targetable nanoassemblies for biomedical utilization.