The great applications of nuclear power for the most promising clean energy sources have been challenged by a large amount of radioactive wastewater generated, specifically the Cs+/Co2+ separation for nuclear waste storage, retreatment or recycling of radioactive wastewater, because of their wide difference in half-life and high heat release. In this work, graphene oxide membranes (GOMs) with interlayer spacing controlled by cations were used to separate mixed Cs+/Co2+ ions. The separation factors of Cs+/Co2+ for K+-controlled graphene oxide membranes (K-GOMs) was 2∼3 times higher than that of GOMs without treatment. In addition, the separation factors of Cs+/Co2+ for K-GOMs can be further enhanced with the increase of membranes thickness and change the initial ratios of the two ions. Typically, the separation factors of K-GOMs with a thickness of ∼300 nm reached up to 73.7 ± 3.9. Moreover, the K-GOM showed outstanding stability of the separation performance under long-term operation within 7 days. First-principles calculation revealed that the enhanced ionic selectivity of controlled GOM is induced by the difference of adsorption energies between the hydrated cations and aromatic rings, resulting in a significant increase in the mobility differences between Cs+ and Co2+ through a fixed narrow interlayer spacing. This study demonstrated excellent separation performances of GO-based membranes based on their size-exclusion effect rather than electrostatic repulsion effect, and we believe this work can enable potential efficient treatment technologies for radioactive wastewater needed urgently.