The advanced glycation end (AGE) product detection based on skin fluorescence spectroscopy is widely used in the screening and evaluation of diabetes and its complications. However, this method is limited by the specific absorption and scattering characteristics in biological tissues and a variety of fluorescent components. In this study, the optimal excitation wavelength combination for measuring skin tissue fluorescence spectra was determined by analyzing the three-dimensional fluorescence spectra of common fluorescent components in human skin tissues. A discrete three-dimensional fluorescence spectrum measurement system was built that integrated the measurement module of tissue diffuse reflectance spectra. The methods of extracting tissue physiological parameters under the diffusion theory and separating discrete three-dimensional fluorescence spectra by multi-peak Gaussian fitting were studied. A clinical community cohort study was then carried out. The results showed that the mean relative concentrations of melanin and deoxyhemoglobin in the diabetic group were lower than those in the normal control group, while the reduced scattering coefficient at 500 nm was higher than that in the normal control group. These differences were statistically significant. No significant difference was found in the relative concentration of oxyhemoglobin. Under the multi-peak Gaussian fitting, 78 fluorescence features were obtained for each subject, and 50 fluorescence features remained after features with insignificant differences (p>0.1) were eliminated. Differential tissue physiological parameters and fluorescence characteristics were pooled, and a diabetes screening model was built by the logistic regression analysis. According to the analysis results of the receiver operating characteristic (ROC) curve, when the proposed method was applied to diabetes screening, the area under the ROC curve of the prediction training set was 0.793, and that of the prediction test set was 0.799. In contrast, that of the single-wavelength skin fluorescence (Sf₃₆₅) was 0.731, which indicated that the proposed model had a better diagnostic value than that of the single-wavelength fluorescence.