Many synthetic microspheres are spherical and symmetrical because of the thermodynamical limitations of the reaction systems. We developed modification techniques to prepare spherical microspheres with intrinsic unsymmetry, for example, a microsphere with both an anionic and a cationic part. The modification was performed at the liquid-solid interface. Reactive microspheres with p-nitrophenyl moieties were settled onto the IgG-preadsorbed substrate, and the reaction between the activated ester moieties and Ige molecules proceeded only at the interface upon attachment. After the reaction, the modified microspheres were detached from the substrate with ultrasonication. In another modification at the air-water interface, the dispersion of reactive microspheres in ethanol was first spread on a water surface to produce the monolayer of microspheres, and then hydrolysis at the water side of the monolayer was performed by adding a NaOH aqueous solution to the subphase. After this unsymmetrical hydrolysis, we carried out IgG immobilization with the remaining reactive moieties. Tn both interface methods, the immobilized IgG molecules were labeled with colloidal gold particles conjugated with anti-IgG antibodies. Transmission electron microscopy indicated that gold particles could be observed on only the immobilized side. Small microspheres were coupled to the reactive microsphere, and its unsymmetry was confirmed by scanning electron microscopy. Such unsymmetrical microspheres will be applicable to the electrical rheology, diagnosis, display technology, and creation of functional devices through the assembling of microspheres.