Molecular assemblies of cationic dipeptide-gold nanoparticle (CDP-AuNP) hybrid microspheres were used to modify cholesterol oxidase electrodes for high-sensitivity cholesterol detection. The cationic dipeptide used here serves as a functional molecule for adsorbing chloroauric acid based on electrostatic interactions and assembly into spherical structures, providing a platform for loading gold nanoparticles (AuNPs), increasing the immobilization load of the enzyme and maintaining the activity of the enzyme as a result of excellent biocompatibility. Moreover, the CDP-AuNP modified cholesterol oxidase electrode has a higher electrocatalytic activity to cholesterol with obvious enhancement in the current response, exhibiting a current response 13 times higher than that of the controlled electrode. The outstanding biocompatibility and increased enzyme load by hybrid microspheres and the good charge-transfer ability of AuNPs in the peptide-based electrode indicate a very attractive perspective in the field of biodevices. (C) 2019 Published by Elsevier Inc.