Single-layered graphene oxide (GO) has exhibited great promise in the areas of sensing, membrane filtration, supercapacitors, bioimaging, and therapeutic carriers because of its biocompatibility, large surface area, and electrochemical, photoluminescent, and optical properties. To elucidate how the physical dimensions of GO affect its intrinsic properties, we employed sonication to produce more than 130 different sizes of GO in aqueous dispersion and implemented new approaches to characterize various GO properties as a function of the average flake size. New protocols were developed to determine and compare the flake size of GO dispersions sonicated with energies up to 20 MJ/g by using dynamic light scattering and atomic force microscopy (AFM). The relationship between the average flake size and sonication energy per unit mass of GO was observed to follow a power law. AFM height measurements showed that the sonication of GO yielded monolayered flakes. Photoluminescence of GO was characterized as a function of the sonication energy (or the average flake size which is the monotonic function of the sonication energy), excitation wavelength, and pH of the dispersion. The strong dependence of the photoluminescence intensity on pH control and the variation of the photoluminescence intensity with different flake sizes were observed. An intense photoluminescence signal, likely related to the separation of the oxidative debris from the GO framework, was found at the highest sonication energies (E ≳ 15 MJ/g) or under extremely alkaline conditions (pH ≳ 11). The cytotoxicity of GO was studied with various flake sizes. Size- and concentration-dependent cytotoxicity was observed for cell lines NIH 3T3 and A549. The NIH 3T3 cell line also demonstrated time-dependent cytotoxicity.