Design of graphene-based materials for biomedical purposes is of great interest. Graphene oxide (GO) represents the most widespread type of graphene material in biological studies. In this work, thin GO sheets were further chemically functionalized with DOTA (1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid), a stable radiometal chelating agent, by an epoxide opening reaction. We report the tissue distribution of the functionalized GO sheets labeled with radioactive indium (111In) after intravenous administration in mice. Whole body single photon emission computed tomography (SPECT/CT) imaging, gamma counting studies, Raman microscopy and histological investigations indicated extensive urinary excretion and predominant spleen accumulation. Intact GO sheets were detected in the urine of injected mice by Raman spectroscopy, high resolution transmission electron microscopy (HR-TEM) and electron diffraction. These results offer a previously unavailable pharmacological understanding on how chemically functionalized GO sheets transport in the blood stream and interact with physiological barriers that will determine their body excretion and tissue accumulation.