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The adsorption of lithium ions(Li⁺) and the separation of lithium isotopes have attracted interests due to their important role in energy storage and nuclear energy, respectively. However, it is still challenging to separate the Li⁺ and its isotopes with high efficiency and selectivity. A novel cellulose-based microsphere containing crown ethers groups (named as MCM-<i>g</i>-AB15C5) was successfully synthesized by pre-irradiation-induced emulsion grafting of glycidyl methacrylate (GMA) and followed by the chemical reaction between the epoxy group of grafted polymer and 4′-aminobenzo-15-crown-5 (AB15C5). By using MCM-<i>g</i>-AB15C5 as adsorbent, the effects of solvent, metal ions, and adsorption temperature on the adsorption uptake of Li⁺ and separation factor of ⁶Li/⁷Li were investigated in detail. Solvent with low polarity, high adsorption temperature in acetonitrile could improve the uptake of Li⁺ and separation factor of lithium isotopes. The MCM-<i>g</i>-AB15C5 exhibited the strongest adsorption affinity to Li⁺ with a separation factor of 1.022 ± 0.002 for ⁶Li/⁷Li in acetonitrile. The adsorption isotherms in acetonitrile is fitted well with the Langmuir model with an ultrahigh adsorption capacity up to 12.9 mg·g⁻¹, indicating the unexpected complexation ratio of 1:2 between MCM-<i>g</i>-AB15C5 and Li⁺. The thermodynamics study confirmed the adsorption process is the endothermic, spontaneous, and chemisorption adsorption. As-prepared novel cellulose-based adsorbents are promising materials for the efficient and selective separation of Li⁺ and its isotopes.