Colloidal aqueous suspension of superparamagnetic nanoparticles (9 nm in diameter) composed of maghemite (gamma Fe2O3) and forming an ionic ferrofluid in aqueous solution are covalently coupled with lectins, enzymes or antibodies, using specific thiol chemistry. The surface charge modifications of nanoparticles, caused by ligand coupling, were monitored by measuring their electrophoretic mobilities using laser-Doppler velocimetry. Particle electrophoretic mobility (PEM) changes are shown to correlate well with the amount of ligand fixed on the particles, as probed by its biological activity. The PEM method provides a useful tool to optimize ligand immobilization at the surface of nanoparticles, and may be advantageous when biological activity measurements are not convenient.