Monodisperse cross-linked polymer microspheres with carboxyl groups on their surface were prepared by means of emulsifier-free emulsion copolymerization. The characterizations by X-ray photoelectron spectroscopy and conductometric titration indicate that most carboxyl groups are located on the surface of microspheres. Their swelling degree or cross-linking degree has been investigated by an in situ swelling method. Moreover, these microspheres can be easily and quickly self-organized into two- or three-dimensional ordered multilayer films or latex crystals.