Small-molecule drugs play an important role in the treatment of various diseases. The screening of enzyme inhibitors is one of the most important means in developing therapeutic drugs. Herein, we demonstrate a liquid crystal (LC)-based screening assay assisted with enzyme catalysis-induced aptamer release for screening xanthine oxidase (XOD) inhibitors. The oxidation of xanthine by XOD prevents the specific binding of xanthine and its aptamer, which induces a bright image of LCs. However, when XOD is inhibited, xanthine specifically binds to the aptamer. Correspondingly, LCs display a dark image. Three compounds are identified as potent XOD inhibitors by screening a small library of triazole derivatives using this method. Molecular docking verifies the occupation of the active site by the inhibitor, which also exhibits excellent biocompatibility to HEK293 cells and HeLa cells. This strategy takes advantages of the unique aptamer-target binding, specific enzymatic reaction, and simple LC-based screening assay, which allows high-throughput and label-free screening of inhibitors with high sensitivity and remarkable accuracy. Overall, this study provides a competent and promising approach to facilitate the screening of enzyme inhibitors using the LC-based assay assisted with the enzyme catalysis-induced aptamer release.