A real-time electrochemical sensor for detecting cholesterol was constructed due to the mimic enzyme characteristics of the metal organic framework (MOF). A new-type composite metal-organic framework (Fe₃O₄@Au/MOF) nanoparticle was prepared by self-assembly method, which had the advantages of recyclability of Fe₃O₄ magnetic particles and accelerating electron transfer of Au nanoparticles. The structure and morphology of the successfully prepared Fe₃O₄@Au/MOF were characterized by infrared spectroscopy, transmission scanning electron microscopy and X-ray photoelectron spectroscopy. With the dual catalytic performance of Fe₃O₄@Au/MOF peroxidase-like enzymes, cholesterol was oxidized to cholestyrtrione and H₂O₂. The oxidatively active hydroxyl radical produced by H₂O₂ can promote oxidation-reduction reaction of 3,3′,5,5′-tetramethylbenzidine (TMB), which increased the movement of charges in the system. According to the response of the electrical signal, the rapid qualitative and efficient quantitative detection of cholesterol can be realized. The research results showed that the optimal detection system determined by cyclic voltammetry was that the reaction temperature of 50℃, the addition amount of Fe₃O₄@Au/MOF of 0.0125g, scanning rate of 0.10V/s and buffer solution pH of 4. The working curve equation was y=20.6401x+4.7722, R² was 0.9956, and the minimum detection limit was 2.7μmol/L, when the cholesterol concentration located in the range of 0.001~1.500mmol/L. The detection system had a good recovery rate and anti-interference with a good application prospects in food analysis and biochemical analysis.