This paper introduced a cholesterol detection method based on hemin chloride mimetics enzyme catalyzed the production of fluorescence by tyramine peroxide. Fluorescence（with excitation/emission maxima at 320 and 426 nm） increased linearly in the 0.1 μmol/L to 2 μmol/L cholesterol concentration range at the optimum conditions. The correlation equation was F=34.85C+165.3 （R2=0.992）. The detection limit based on the 3σ/k criterion reached 0.08 μmol/L. The proposed assay was successfully employed for cholesterol detection in （spiked） several kinds of milk samples with recoveries of 98.7%-112.7%. Given its high sensitivity and good stability， this method has good specificity through selective decomposition of the substrate by cholesterol oxidase. Therefore， the method can be used to detect cholesterol in milk.