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Superparamagnetic iron oxide nanoparticles (SPIONs) have attract a great deal of interest in biomedical research and clinical applications over the past decades. Taking advantage the fact that SPIONs only exhibit magnetic properties in the presence of an applied magnetic field, they have been used in both in vitro magnetic separation and in vivo applications such as hyperthermia (HT), magnetic drug targeting (MDT), magnetic resonance imaging (MRI), gene delivery (GD) and nanomedicine. Successful applications of SPIONs rely on precise control of the particle's shape, size, and size distribution and several synthetic routes for preparing SPIONs have been explored. Tailored surface properties specifically designed for cell targeting are often required, although the generic strategy involves creating biocompatible polymeric or non-polymeric coating and subsequent conjugation of bioactive molecules. In this review article, synthetic routes, surface modification and functionaliztion of SPIONs, as well as the major biomedical applications are summarized, with emphasis on in vivo applications.