The separation of benzene derivatives is energy intensive and laborious as a result of the overlapping physicochemical properties of these isomers. Here, we report on the separation of ortho-disubstituted benzene isomers using cucurbituril (CB7) aqueous solution with more than 92% selectivity. Thermodynamic and kinetic analysis proves that the ortho-isomer has stronger binding ability and slower decomplexation rate constant than the para- and meta-isomers when hosted by CB7. Optimized host-guest models indicate that the ortho-isomer with the smallest aspect ratio well matches the spherical interior cavity of CB7, resulting in highly stable complexes. Furthermore, laboratory scale-up experiments using commercial xylenes and C8 aromatic fraction of pyrolysis gasoline proved that CB7 is able to separate ortho-xylene (OX) with a remarkable selectivity of up to 83%. We believe that this work accentuates the role of molecular recognition studies using macrocyclic hosts to improve the quality and energy bill of critical industrial separations. Video Abstract:[Figure presented] Disubstituted benzene isomers are widely used as starting materials or solvents in the petrochemical and pharmaceutical industries. Selective separation of these isomers has been one of the most challenging topics because of their similar physicochemical properties. Here, we present a unique strategy for separating and purifying ortho-disubstituted benzene compounds with different functional groups from their corresponding isomers by the highly stable (chemical, moisture, and thermal) macrocyclic host cucurbituril (CB7). The separation process can be performed under ambient temperature and pressure with more than 92% selectivity after one extraction cycle. This research demonstrates that macrocyclic hosts such as CB7 can be used as ideal porous materials for facile and economical protocols in energy-intensive chemical separations. Selective separation of substituted benzene isomers bearing a diverse range of functional groups is a pressing challenge in the chemical industry. In this article, we present a liquid-liquid extraction method that can efficiently separate four kinds of ortho-disubstituted benzene molecules from their isomers by using the highly stable (chemical, moisture, and thermal) macrocyclic host cucurbituril (CB7). This host-guest separation process can be performed under ambient temperature and pressure with more than 92% selectivity after one extraction cycle.