Abstract The detection of α-glucosidase (AGLU) inhibitors is critical for the screening of anti-diabetic drugs. In this study, we first demonstrate a liquid crystal (LC)-based assay to detect anti-diabetic drugs. When the solution of non-ionic surfactant dodecyl α-D-glucopyranoside (DDG) is introduced onto the LCs, self-assembled monolayers are formed at the aqueous/LC interface, which induces the perpendicular orientation of LC molecules at the interface. Accordingly, the LCs show a dark image. However, when a mixture of AGLU and DDG is introduced onto the LCs, a bright image is observed due to enzymatic hydrolysis of DDG by AGLU, which prevents formation of the surfactant monolayers and results in the planar or tilted orientation of LC molecules at the interface. Using the LC-based AGLU assay, the detection of three AGLU inhibitors that are popular anti-diabetic drugs including acarbose, migliol, and voglibose is demonstrated. In addition, the linear detection ranges and the detection limits of these drugs are also determined. This method provides a simple and powerful strategy to rapidly and accurately detect AGLU inhibitors, which is very promising in the applications of screening anti-diabetic drugs.