BACKGROUND: Magnetic nanoparticles are of immense interest for their applications in biotechnology. This paper reports the synthesis of magnetic iron oxide nanoparticles from two different water-in-oil microemulsion systems (ME-MIONs), their characterization and also their use in purification of coagulant protein. RESULTS: ME-MIONs have demonstrated to be an efficient binder in the purification of Moringa oleifera protein when compared with the superparamagnetic iron oxide nanoparticles prepared from coprecipitation in aqueous media. The size and morphology of the ME-MIONs were studied by transmission electron microscopy (TEM) while the structural characteristics were studied by X-ray diffraction (XRD). The microemulsion magnetic iron oxide nanoparticles (ME 1-MION and ME 2-MION) obtained have a size range 7-10 nm. The protein and ME-MIONs interaction was investigated by Fourier transform infrared spectroscopy (FT-IR); the presence of three peaks at 2970, 2910 and 2870 cm(-1) respectively, confirms the binding of the protein. The purification and molecular weight of the coagulant protein was 6.5 kDa as analyzed by SDS-PAGE. CONCLUSION: The ME-MIONs have the advantage of being easily tailored in size, are highly efficient as well as magnetic, cost effective and versatile; they are, thus, very suitable for use in a novel purification technique for protein or biomolecules that possess similar characteristics to the Moringa oleifera coagulant protein. (C) 2011 Society of Chemical Industry