Magnetic iron oxide nanoparticles (MNPs) have been developed and applied for a broad range of biomedical applications, such as diagnostic imaging, magnetic fluid hyperthermia, targeted drug delivery, gene therapy and tissue repair. As one key element, reproducible synthesis routes of MNPs are capable of controlling and adjusting structure, size, shape and magnetic properties are mandatory. In this review, we discuss advanced methods for engineering and utilizing MNPs, such as continuous synthesis approaches using microtechnologies and the biosynthesis of magnetosomes, biotechnological synthesized iron oxide nanoparticles from bacteria. We compare the technologies and resulting MNPs with conventional synthetic routes. Prominent biomedical applications of the MNPs such as diagnostic imaging, magnetic fluid hyperthermia, targeted drug delivery and magnetic actuation in micro/nanorobots will be presented.