Abstract: The level of cholesterol in human serum acts as one of the important indicators for clinical diagnosis and health management. A ratiometric fluorescence sensor was constructed for cholesterol detection, in which mercapto-β-cyclodextrin-gold nanoclusters were used as sensing probes and rhodamine B as reference probes. Gold nanoclusters and rhodamine B dye were co-assembled on a dendritic mesoporous silica substrate with electrostatic interaction. Based on the enhanced fluorescence of gold nanoclusters caused by the host-guest recognition between the cyclodextrin cavity and cholesterol, the determination of serum cholesterol is realized. Experimental results show that the highest detection sensitivity is obtained when the reaction time is 1 min, the media pH value is 6.0 and the reaction temperature is 28 ℃. The calibration curve can be expressed as F₂/F₁= 0.0209 C+ 1.4490 in the concentration range of 5.00 ～ 50.00 μmol·L⁻¹. Common coexisting components in the serum did not interfere with the detection of cholesterol. The ratiometric probe obviates the interference of background fluorescence in a complex biological matrix. The method provides a sensitive and rapid determination of cholesterol.