Adsorptive membrane-based chromatography can provide the high separation efficiency common to column chromatography but at a lower working pressure. Herein, a novel membrane chromatography system for lithium isotope adsorptive separation is reported. It uses polysulfone-graft-4\'-aminobenzo-15-crown-5-ether (PSf-g-AB15C5) porous membranes (0.52 mmol/g of immobilization crown ether, average pore size of 62.7 nm, porosity of 80.4%) as a stationary phase packed in a chromatography column (Ø 25 × 100 mm). Furthermore, a four-stage tandem membrane chromatography system was designed to enhance lithium isotope separation performance. The partial eluate from the former column was used as the feed solution for the next stage. Results show that the flow rate of the eluent could reach 18 mL/h owing to the lower internal diffusion resistance of membranes. Meanwhile, adsorption isotherms and adsorption kinetics show that Li+ adsorption was an exothermic and spontaneous process. The surface diffusion, multilayer adsorption and ion-pore electrostatic interaction between Li+ and the crown ether groups on the membranes played a key role in the separation of 7Li+ and 6Li+ by membrane chromatography. The separation factor obtained from the single-stage membrane chromatography was up to 1.0232. The abundances of 7Li+ and 6Li+ gradually increased with an increase in the elution stages. The relative abundances of 7Li+ and 6Li+ obtained from the four-stage tandem membrane chromatography increased by 0.26% (from 92.40 to 92.66%) and 0.2% (from 7.60 to 7.80%), respectively. In conclusion, our current research opens a new avenue for the simultaneous enrichment of 7Li+ and 6Li+ during lithium isotope adsorptive separation.