Carbon-based nanomaterials functionalized with fluorescent and water-soluble groups have emerged as platforms for biological imaging because of their low toxicity and ability to be internalized by cells. The development of imaging probes based on carbon nanomaterials for biomedical studies requires the understanding of their biological response as well as the efficient and safety exposition of the nanomaterial to the cell compartment where it is designed to operate. Here, we present a fluorescent probe based on surface functionalized carbon nano-onions (CNOs) for biological imaging. The modification of CNOs by chemical oxidation of the defects on the outer shell of these carbon nanoparticles results in an extensive surface functionalization with carboxyl groups. We have obtained fluorescently labelled CNOs by a reaction involving the amide bond formation between fluoresceinamine and the carboxylic acids groups on the surface of the CNOs. The functionalized CNOs display high emission properties and dispersability in water due to the presence of high surface coverage of carboxylic acid groups that translate in an efficient fluorescent probe for in vitro imaging of HeLa cells, without significant cytotoxicity. The resulting nanomaterial represents a promising platform for biological imaging applications due to the high dispersability in water, its efficient internalization by cancer cells and localization in specific cell compartments.