Cholesterol is an important lipid present in animal cells and is involved in the synthesis of various hormones and vitamins. As excess cholesterol in human serum can cause various health issues, the selective detection of serum cholesterol is critical for medical diagnosis and treatment. However, enzyme-based detection is commonly used to achieve the detection selectivity. Here, we report a molecular-imprinted nanocomposite for fluorescence-based detection of cholesterol without using any enzymes. The nanocomposite is composed of polycyclodextrin and graphene oxide with the imprint of cholesterol. Rhodamine 6G binds with the cyclodextrin component of this composite via host–guest interactions, and the graphene oxide component quenches its fluorescence. The addition of cholesterol triggers the displacement of rhodamine 6G and recovers the fluorescence. The nanocomposite shows high specificity toward cholesterol with a detection sensitivity in the micromolar range. This material has been used for capillary-based visual detection of cholesterol and color-scanning mobile-app-based rapid diagnosis of cholesterol in human serum. Developed materials and methods can be used as alternatives of commonly used enzyme-based detection approaches.