Graphite oxide was synthesized by the oxidation of graphite via a modified Hummers approach. The oxygen content of graphite oxide was tuned by changing the oxidation time. The saturated ratio of oxygen to carbon is 0.47. Furthermore, the effect of oxygen content on the structures of graphite oxide was evaluated by using SEM, TEM, XRD, Raman, FTIR, TG, TPD-MS, and nitrogen adsorption. It was found that the types of functional groups in graphite oxides, which are epoxy, hydroxyl, and carboxyl groups, were independent of oxygen content. However, the interplanar space and BET surface area of graphite oxide linearly increased with increasing O/C ratio. Furthermore, the structure of graphite can be easily and remarkably disordered even by partial oxidation.