Molecular imprinting was conducted on a thin polymeric membrane stacked on the self-assembled monolayer of a gold plate. The covalent bonding method was used in the molecular imprinting process. Cholesterol was used as a target molecule. Coating with poly(methyl methacrylate) was followed by hydrolysis and extraction of cholesterol. The remaining site was used to recognize cholesterol. Using the cholesterol-imprinted gold electrode as a working electrode, the cholesterol recognition ability was estimated. Various cholesterol analogs were used to obtain the comparative data to determine the imprinting and selectivity factors. The results showed that molecular similarity was important to enhance the recognition. However, the presence of a hydroxyl functional group played a key role in enhancing the molecular recognition, suggesting that molecular polarity and hydrophobicity are important factors.