Understanding the interactions of graphene oxide (GO)-based materials with biological systems is critical due to the materials’ potential applications. Herein, we investigated the extent to which singe-to-few-layer GO sheets of different controlled lateral dimensions, translocate from the nose to the brain following intranasal instillation. We explore tissue location and in vivo biodegradability of the translocated materials using various techniques. Mass spectrometry- and confocal Raman-based analysis, indicated that trace amounts of administered GO underwent nose-to-brain translocation in a size-dependent manner. The smallest GO-sheet size category [ultra-small (us)-GO, 30–500 nm] gained greatest access to the brain in terms of quantity and coverage. Confocal Raman mapping and immunofluorescence combinations showed that in vivo us-GO sheets resided in association with microglia; Raman spectroscopy showed that trace quantities of us-GO were maintained over a month, but underwent biodegradation-related changes. This study adds to growing awareness regarding the fate of graphene-based materials in biological systems.