For biomedical application, the functionalization of the COOH groups and the concentration evalutation of COOH groups on the Fe₃O₄ nanoparticles play important roles. Fe₃O₄ nanoparticles with carboxyl functional groups have been successfully fabricated by a simple and easy-to-implement chemical method, through the combination of Fe₃O₄ nanoparticles with silane complexes containing COOH radicals. This complex is the product of the reaction between succinic anhydride (SA) and 3-aminopropyltriethoxysilane (APTES). The fabricated Fe₃O₄-COOH nanoparticles have superparamagnetic properties with high technical saturation magnetization (~22 emu/g), and the concentration of carboxyl functional groups on the surface of Fe₃O₄ nanoparticles has been quantified based on the method using methylene blue indicator (MB) through ultraviolet-visible absorption spectroscopy (UV-vis).