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A simple and sensitive electrochemical cholesterol biosensor was fabricated based on ceramic-coated liposome (cerasome) and graphene quantum dots (GQDs) with good conductivity. The cerasome consists of a lipid-bilayer membrane and a ceramic surface as a soft biomimetic interface, and the mild layer-by-layer self-assembled method as the immobilization strategy on the surface of the modified electrode was used, which can provide good biocompatibility to maintain the biological activity of cholesterol oxidase (ChOx). The GQDs promoted electron transport between the enzyme and the electrode more effectively. The structure of the cerasome-forming lipid was characterized by Fourier transform infrared (FT-IR). The morphology and characteristics of the cerasome and GQDs were characterized by transmission electron microscopy (TEM), zeta potential, photoluminescence spectra (PL), etc. The proposed biosensors revealed excellent catalytic performance to cholesterol with a linear concentration range of 16.0 × 10−6–6.186 × 10−3 mol/L, with a low detection limit (LOD) of 5.0 × 10−6 mol/L. The Michaelis–Menten constant (Km) of ChOx was 5.46 mmol/L, indicating that the immobilized ChOx on the PEI/GQDs/PEI/cerasome–modified electrode has a good affinity to cholesterol. Moreover, the as-fabricated electrochemical biosensor exhibited good stability, anti-interference ability, and practical application for cholesterol detection. [ABSTRACT FROM AUTHOR]