Determining cholesterol levels is of great importance in the detection of high risk for heart disease. Here, we developed a real-time sensing strategy for free cholesterol molecules, employing ultraviolet (UV)-treated liquid crystals (LCs). When 4-cyano-4'-pentylbiphenyl (5CB), a nematic LC material, is treated with UV light, it shows a bright-to-dark optical response to a very small change in pH from 7.0 to 7.1, which can be observed with a polarizing optical microscopy at the aqueous/LC interface. In diagnostic applications, the second most widely used enzymatic reaction between cholesterol and cholesterol oxidase is able to generate H+, which could be detected by pH-sensitive LCs. Exploiting these mechanisms, a cholesterol sensor was designed and the device performed well in a range of cholesterol concentrations from 10 to 300 mg/mL, which covers the physiologically relevant range of cholesterol in the human body (90-220 mg/mL). We also determined the limit of detection, 1 x 10(-9) mg/mL, and explored the effects of inhibitors on this sensor. The results obtained from this study may offer a simple tool for the detection of cholesterol compared with conventional methods and have potential for clinical diagnostics.