Here, we demonstrate use of acetylcholinesterase (AChE)-responsive polyoxometalate (POM)/surfactant supramolecular spheres to build a liquid crystal (LC)-based sensing platform for detection of organophosphorus pesticides. The self-assembled spheres are composed of hybrid materials of a POM, sodium dodecatungstophosphate (PW12), and a surfactant, myristoylcholine (Myr). It displays dark appearance when the aqueous solution is in contact with LCs supported on the octadecyltrichlorosilane-treated glass deposited with the supramolecular spheres, suggesting perpendicular orientation of LCs at the aqueous/LC interface. In contrast, LCs show bright appearance when the surface-deposited supramolecular spheres are enzymatically hydrolyzed by AChE, corresponding to planar orientation of LCs at the aqueous/LC interface. Detection of organophosphates are successfully achieved as they are potent inhibitors of AChE. The detection limit of the sensing platform reached 0.9 ng/mL for dimethoate. This method can avoid disturbance of external interference with excellent specificity and sensitivity, which makes it very promise in detection of organophosphorus pesticides.