Skin cholesterol is a novel biomarker to assess the risk of atherosclerotic diseases. A system based on diffuse reflectance spectroscopy technology is designed for noninvasive and rapid detection of skin cholesterol. The feasibility of the system is validated through detecting digitonin-horseradish peroxidase copolymer solution which simulates the skin cholesterol of gradient concentration and skin cholesterol in vivo of subjects. A parameter S based on the relative diffuse reflectance increases linearly with the increasing concentration of the copolymer solution in the wavelength band from 440 nm to 550 nm (correlation coefficient r=0.992, P<0.01). After adjusting age, gender and other factors, it shows significant positive correlation between the skin cholesterol test results of subjects and the total cholesterol, low density lipoprotein cholesterol, and the correlation coefficients are 0.837 (P<0.01) and 0.778 (P<0.01), respectively. The diffuse reflectance spectroscopy is a noninvasive and convenient method for the detection of skin cholesterol, and the noninvasive detection of skin cholesterol in vivo contributes to the early detection of atherosclerotic diseases.