We propose a novel method for the ultrarapid cationization of gold nanoparticles with diameters ranging from several to a hundred nanometers via a single-step ligand exchange reaction of citrate-protected anionic gold nanoparticles with cationic thiol ligand. This reaction was performed only in an aqueous medium via a single step from citrate to cationic thiol and therefore enables a rapid preparation of cationic Au nanoparticles without a contamination of organic solvent or lipid-soluble molecules. The cationization was successfully completed within 20 min without changes in the core diameter and optical characteristic.