Separation and purification of graphene oxide (GO) prepared from chemical oxidation of flake graphite and ultrasonication by capillary electrophoresis (CE) was demonstrated. CE showed the ability to provide high-resolution separations of GO fractionations with baseline separation. The GO fractionations after CE were collected for Raman spectroscopy, atomic force microscopy, and transmission electron microscopy characterizations. GO nanoparticles (unexfoliated GO) or stacked GO sheets migrated toward the anode, while the thin-layer GO sheets migrated toward the cathode. Therefore, CE has to be performed twice with a reversed electric field to achieve a full separation of GO. This separation method was suggested to be based on the surface charge of the GO sheets, and a separation model was proposed. This study might be valuable for fabrication of GO or graphene micro- or nanodevices with controlled thickness.