Small carbon nano-onions (CNOs, 6–8 shells) were prepared in high yield and functionalized with carboxylic groups by chemical oxidation. After functionalization these nanostructures were soluble in aqueous solutions. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 tetrazolium (MTS) tests showed excellent cytocompatibility of all CNOs analyzed at 30 and 300 μg mL−1, so these carbon nanostructures can be safely used for biological applications. The first covalent functionalization of oxidized CNOs (ox-CNOs) with biomolecules, by using biotin–avidin interactions is reported here. Multilayers were prepared on a gold surface by layer-by-layer assembly and the process was monitored by surface plasmon resonance (SPR) spectroscopy and atomic force microscopy (AFM). Covalent binding of molecules to the short amine-terminated organosulfur monolayers was assessed by Fourier transform infrared spectroscopy using total attenuated reflactance mode (FT-IR/HATR).