Separating xylene isomers is a challenging task due to their similar physical and chemical properties. In this study, we developed a molecular sieve incorporating a reduced graphene oxide (rGO) membrane for the precise differentiation of xylene isomers. We fabricated GO membranes using a vacuum filtration technique followed by thermal-induced reduction to produce rGO membranes with precisely controllable interlayer spacing. Notably, we could finely tune the interlayer spacing of the rGO membrane from 8.0 to 5.0 Å by simply varying the thermal reduction temperature. We investigated the reverse osmosis separation ability of the rGO membranes for xylene isomers and found that the rGO membrane with an interlayer spacing of 6.1 Å showed a high single component permeance of 0.17 and 0.04 L m −2 h −1 bar −1 for para-and ortho-xylene, respectively, exhibiting clear permselectivity. The separation factor reached 3.4 and 2.8 when 90:10 and 50:50 feed mixtures were used, respectively, with permeance 1 order of magnitude higher than that of current state-of-the-art reverse osmosis membranes. Additionally, the membrane showed negligible permeance and selectivity decay even after continuous operation for more than 5 days, suggesting commendable membrane resistance to solvent swelling and operating pressure.