In this study, we report on a label-free biosensor for the detection of acetylcholine (ACh) and an acetylcholinesterase (AChE) inhibitor, utilizing liquid crystals (LCs) on a polymeric surface with periodic nanostructures. The polymeric nanostructures, which hold sinusoidal anisotropic patterns, were fabricated by a sequential process of poly(dimethylsiloxane) buckling and replication of these patterns on a poly(urethane acrylate) surface where a film of gold was deposited. AChE was then covalently immobilized onto the gold surface. Optical images of nematic 4-cyano-4′-pentylbiphenyl (5CB) revealed that it aligned parallel to the plane of the enzymedecorated surface. However, AChE-mediated hydrolysis of ACh resulted in a plume of choline, acetate, and unreacted ACh, which in turn produced a distinctive optical transition of 5CB (from uniform to random) by masking the anisotropic surface topography. The hydrolysis of ACh was inhibited in the presence of a carbamate insecticide (AChE inhibitor), which prevented the orientational transition of 5CB by decreasing the enzymatic activity of AChE. These results suggest that this LC-based enzymatic sensor is highly sensitive to ACh and the AChE inhibitor, with a detection limit of 10 and 1 nM, respectively. This study demonstrates that polymeric surfaces with continuous wavy features can be used as LCbased biosensors to amplify the existence of ACh and an AChE inhibitor without labeling or an additional amplification strategy.