The separation of Th(IV)/U(VI) is of great importance in the development of thorium-based reactors. Here, we report our studies of Th(IV)/U(VI) separation by graphene oxide (GO). Firstly, variation of media conditions such as the pH, contact time, ionic strength, as well as the presence of different anionic ligands were conducted. The results showed that GO exhibited great selectivity for Th(IV) and the separation factor of Th(IV)/U(VI) reached as high as 36.3 after 2. h of contact at pH 3.8 and minimum ionic strength, and increasing the ionic strength caused the separation factor of Th(IV)/U(VI) to decrease. The presence of sulfate strongly influenced the separation factor of Th(IV)/U(VI) by GO. Secondly, competitive adsorption studies, as well as SEM-EDS, XRD, FT-IR, and XPS characterization were employed to gain further understanding of the mechanism of GO's selective complexation of Th(IV)/U(VI). FT-IR and XPS characterizations showed that GO adsorbed Th(IV)/U(VI) through coordination of functional groups such as C. O and C. O on GO. The results of the competitive adsorption studies suggested that GO had a selective affinity for Th(IV) over U(VI). SEM-EDS exhibited a uniform distribution of both Th(IV) and U(VI) on GO. XRD indicated that the interlayer distances of the prepared GO, GO-Th(IV), GO-U(VI), and GO-Th(IV)/U(VI) were 8.24, 8.20, 7.86, and 8.26. Å, respectively. These results suggested that the adsorption of U(VI) caused a decrease of the interlayer distance of GO, which is probably inhibits GO's ability to adsorb metal ions due to increased inter-planar repulsion and induced steric hindrance that limits adsorption of more metal ions. Therefore, a spatial feature based mechanism of GO was proposed.