Graphene oxide nanosheets often bear a wide size distribution. However, it is critical to have nanosheets with narrow size distribution for their unique size-dependent physiochemical properties, and nanosheets with a narrow size distribution are the cornerstones for application. Therefore, efficient separation methods of graphene nanosheets have been given considerable attention in many scientific areas recently. Free-flow electrophoresis is extensively used in the separation and purification of biological molecules with continuous flow separation. The charged graphene oxide nanosheets to some extent are very close in size to biological molecules and share similarity in motion behavior in an electric field. Thus, in the present work, we present a new and simple means to separate graphene oxide nanosheets into more mono-dispersed size groups by using the free-flow electrophoresis technique. By optimizing the separation conditions, we were able to obtain graphene oxide sheets with narrow size distribution. The separated samples were characterized by atomic force microscopy, and the size measurements were made by using the software "Image Pro Plus." In addition, a brief discussion is also given into the theoretic background of the separation of graphene oxide according to the size by the technique of preparative free-flow electrophoresis.