Due to the urgent need for noninvasive detection of skin cholesterol, a portable, intelligent and real-time skin diffuse reflectance spectroscopy measurement system was designed based on a micro-spectrometer. Digitonin-horseradish peroxidase copolymer solution was prepared. According to the properties digitonin binds to the hydroxy of cholesterol molecular specifically and the horseradish peroxidase reacts with TMB color solution (the main component is 3,3’,5,5’-tetramethylbenzidine ) a color change was produced, by which the skin cholesterol was identified and instructed with high sensitivity and high specificity, and the concentration of skin cholesterol was quantified by measuring the degree of color change. In order to validate the feasibility of this method, pig skin which is similar to human skin was taken as the experimental subject, and cholesterol samples of gradient concentration were achieved through the extraction. After that the spectroscopy measurement system was adopted to detect the cholesterol concentration. The experiment result showed that, relative diffuse reflectance can distinguish the cholesterol samples with different concentrations, and the diffuse reflectance intensity factor can quantity the concentrations of cholesterol at characteristic wavelengths (442, 450 and 463 nm) and characteristic wavelength band of 442~500 nm. Linear fitting curves were obtained with the determination coefficient R2 were 0.960, 0.959, 0.958 and 0.958, respectively. The study has shown that, using diffuse reflectance spectroscopy technology can realize noninvasive rapid detection of skin cholesterol, and applying it to the risk assessment of atherosclerotic diseases would contribute to the prevention and control of such diseases significantly.