We report a novel approach based on non-covalent interactions for the functionalization of carbon nano-onions (CNOs) with fluorophores. , We report a novel approach based on non-covalent interactions for the functionalization of carbon nano-onions (CNOs) with fluorophores. The assembly of pyrene–BODIPY conjugates on the CNO surface by means of π–π-stacking results in fluorescent carbon nanoparticles that are successfully uptaken by HeLa cancer cells with no cytotoxicity observed. The ability to functionalize carbon-based nanomaterials by using mild conditions will pave the way for future clinical application of these versatile nanomaterials.