Graphite oxide samples with different oxidation levels were prepared by the oxidation of natural flake graphite based on a modified Hummers method, using different reaction temperatures and oxidation times followed by ultrasonication then centrifugation to obtain the corresponding graphene oxide nanosheet suspensions. The samples were characterized by XRD and AFM. The results show that the oxidation level of graphite oxide samples can be increased by increasing either the reaction temperature or the oxidation time. Well-dispersed suspensions of the graphite oxide samples can be formed in alkaline solution after ultrasonication, especially for samples with higher oxidation levels. A number of coarse particles are observed in the suspensions, particularly those derived from graphite oxide samples with lower oxidation levels, due to the lower degree of exfoliation in these samples, which is influenced by the oxidation level. Rapid sedimentation of these coarse particles can be achieved by high-speed centrifugation, yielding homogenous suspensions of graphite oxide comprising a mass of monolayer graphene oxide nanosheets with lateral dimensions of several hundred nanometers to several micrometers and thickness of ~1.0–1.4 nm.