In this study, we developed a simple and label-free liquid crystal (LC)-based biosensor for real-time screening of the presence of urease in aqueous solutions. Nematic liquid crystal, 4-cyano-4'-pentylbiphenyl (5CB), when treated with ultraviolet light, showed a bright to dark optical response to the emergence of ammonia in the aqueous phase, indicating a planar to homeotropic alignment of LC. The ammonia-driven optical signal could be ascribed to the orientational transition of 5CB, caused by the deprotonation of 4-cyano-4'-biphenylcarboxylic acid (CBA), which was the main product of UV-treatment and the screening of the electrostatic interactions at the aqueous/LC interface. Due to the high spatial resolution and fast response, this method was successfully used to detect urease belonging to the family of amidohydrolase, which hydrolyzes urea into ammonia and carbon dioxide. A urease concentration as low as 1 nM could be detected and monitored in real-time. Moreover, no signal was detected when a divalent copper ion, which blocks the active sites of urease, was used. This result further confirmed our initial hypothesis that orientational transitions of LC were induced from the enzymatic reaction. The results reported in this article suggest that our method may potentially have clinical utility for the specific detection of Helicobacter pylori. In addition, this ammonia-based LC biosensor may be used for the sensitive detection of other amidohydrolases.