Carbon nano-onions (CNOs) are a versatile class of carbon nanomaterials that recently aroused great interest in the scientific community. Several production methods have been published, where thermal annealing of detonation nanodiamonds (DNDs) is the elected method that ensures high yields along with a low cost. Thanks to their peculiar properties and to the possibilities for functionalizing their surface, CNOs are employed in different fields. In particular, fluorescently labeled CNOs are promising candidates in biomedical applications, such as scaffolds for cellular targeting, imaging, and drug delivery due to their good biocompatibility both in vitro and in vivo. In this chapter, we will present the unique properties of CNOs and the different methods used for their synthesis. Additionally, we will review the potential biological applications of CNOs, and their in vitro and in vivo biosafety assessments.