Controllable synthesis of graphite oxide (GO) for targeted surface properties is of great importance for its versatile applications. For this purpose, GO samples were prepared with different amounts of oxidant and characterized by X-ray diffraction, Fourier transform infrared spectroscopy, C-13 cross-polarization with total sideband suppression magic-angle-spinning nuclear magnetic resonance, X-ray photoelectron spectroscopy, Raman spectroscopy, and zeta-potential measurement. When the oxidant amount is below a critical value, the epoxy groups are dominant on the GO surfaces, together with a few hydroxyl and carbonyl groups. Further increase in the oxidant amount leads to the formation and development of the carboxyl groups, which eventually reach a saturation level. Meanwhile, the increasing oxygen-containing groups introduce more defects and reduce the crystalline graphene domains on GO. A possible mechanism for the formation of the oxygen-containing groups on GO is proposed, providing a guideline for the manipulation of the GO surface properties.