Efficient and energy-saving separation of benzene isomers bearing a diverse range of functional groups is a great challenge due to their overlapping physicochemical properties. Here, we report a water soluble pillararene (WP5) that is successfully used as a multifunctional material for the separation and detection of meta/orthosubstituted benzene isomers in water. A liquid-liquid extraction strategy was used for the separation of these benzene isomers based on their different affinity to WP5 in water. The selectivity for the metaover the ortho- isomer for xylenes, chlorotoluene and bromotoluene was 88.6%, 88.3% and 95.0% respectively, in one extraction cycle. Furthermore, a fluorescence indicator system based on WP5 and a fluorescent dye molecule (10-methylacridinium, D) was adopted and exhibited significant fluorescence and optical discrimination upon the addition of MX compared to OX, which implies that a simple “turn-on” detection can be performed prior to engaging in the separation process.