The biomedical applications of carbon nanomaterials are under intensive investigation for the development of next-generation therapeutics. Although much focus has been placed on carbon nanotubes (CNTs) and graphene, other carbon nanomaterials including carbon nanohorns (CNHs), nanodiamonds (NDs) and fullerenes have emerged as suitable candidates for biomedical applications. Among these multi-shell fullerenes, also known as carbon nano-onions (CNOs), are the less studied carbon nanomaterials in biomedicine. The unique properties of carbon nano-onions, such as high surface area-to-volume ratio, thermal conductivity, electrical conductance, mechanical stiffness and ease of chemical functionalization render them fascinating materials for diverse applications including drug-delivery, diagnostics, biological imaging and tissue engineering. Carbon nanomaterials are emerging as smart nanostructures for biomedicine due to the possibility to incorporate multiple functionalities and moieties internally or externally. They can be modified at a precise physicochemical level to optimize targeting in the complex in vivo environment and also engineered for fluorescence detection, magnetic resonance imaging and ablation of tumor cells. Herein, robust and versatile synthetic strategies for the modification of carbon nano-onions (CNOs) are reported. The development of novel CNO conjugates represent a promising platform for the realization of novel technology scaffold for molecular imaging, photodynamic therapy and molecular transporter of fully synthetic carbohydrate-based vaccines for immunotherapy due to the large specific surface area and unique optical and electrochemical properties of CNOs. Through the methodologies described, these smart nano-materials can envisage the realization of multi stimuli-responsive and dynamic architectures capable of changing their physicochemical behavior upon encountering specific microenvironmental signals becoming relevant for diagnosis, imaging and therapies of specific disease applications.