Nanotechnology has generated tremendous hopes in recent years toward the design of advanced functional materials, especially in the bio-medical field. Nano-sized-materials such as magnetite nanoparticles display indeed fascinating physico-chemical properties that, if tuned properly, can be exploited to design new bio-diagnostic and therapeutic strategies as well as innovative biotechnology methodologies. Owing to their biocompatibility and excellent magnetic properties, magnetite nanocrystals have been the object of a tremendous amount of research in the last decade and numerous (bio)applications have been reported. Importantly, advances in the synthesis of magnetite nanoparticles enable excellent control over their size, shape, and composition. Despite these remarkable progresses, many issues remain to be overcome for these nanotechnology products to revolution the medical practice. The fine control and application of colloidal nanostructures such as magnetite nanoparticles in complex biological systems remains especially challenging. This article attempts to review the current status of magnetite nanoparticles preparation and use, with a special emphasis on bio-medical applications, but also to outline the promises and challenges associated to this emerging technology.