Graphene oxide (GO) is an increasingly important nanomaterial, which exhibits great promise in the area of bionanotechnology and nanobiomedicine. In this study, we synthesized uniform ultrasmall graphene oxide nanosheets with high yield by a convenient way of modified Hummers' method. The uniform ultrasmall GO nanosheets, which exhibit fluorescence property and outstanding stability in a wide range of pH values, were less than 50 nm. Furthermore, because of the advantages of its lateral size, the uniform ultrasmall GO nanosheets showed excellent biocompatibility of lower cytotoxicity and higher cellular uptake amount compared to the random large GO nanosheets. Therefore, the as-prepared uniform ultrasmall GO nanosheets could be explored as the ideal nanocarriers for drug delivery and intracellular fluorescent nanoprobe.