A liquid crystal (LC)-based pH sensor for real-time monitoring of changes in localized pH values near a solid surface is reported, along with its application for the detection of enzymatic activities. It is found that 4-cyano-4′-pentylbiphenyl (5CB), when doped with 4′-pentyl-biphenyl-4-carboxylic acid (PBA), shows a bright-to-dark optical response to a very small change in pH (from 6.9 to 7.0). The pH-driven optical response can be explained by using orientational transitions of 5CB induced by the protonation and deprotonation of PBA at the aqueous/LC interface. Because of its high pH sensitivity, the LC-based sensor is further exploited for monitoring local pH changes resulting from enzymatic reactions. As a proof of concept, the hydrolysis of penicillin G by surface-immobilized penicillinase is monitored using the system, even when the concentration of penicillin G is as low as 1 nM. This type of LC-based sensor may find potential utilities in high-throughput screening of enzyme substrates and enzyme inhibitors.