Advanced glycation end products (AGEs) are a kind of compounds with various structures. When the blood sugar is higher than a normal value, it will be produced in large quantities and cannot be metabolized by the body’s own metabolism, and has a memory function of long-term abnormal blood sugar. Studies have shown that AGEs is one of the important factors causing diabetes and its complications. By detecting the accumulation of AGEs in vivo, the occurrence and development of diabetes and its complications can be predicted. The existing in vitro AGEs detection methods have problems of complicated operation, long detection time, high cost and difficulty in promotion; The in vivo AGEs detection methods have problems such as skin pigmentation, age, and hemoglobin interference. Therefore, based on the good optical properties of the cornea and the autofluorescence characteristics of AGEs, a fluorescence spectroscopic detection method for advanced glycation end products of the cornea was proposed. A set of corneal AGEs fluorescence spectrum detection system was constructed. The system consisted of microfiber spectrometer, integrated LED excitation light source, Y-type 12+1 fiber and PC-side spectral processing display software. The fluorescence spectrum detection system was used to collect data from 17 volunteers (9 males, 8 females, 4 diabetics, the youngest 15 years old and the oldest 81 years old) in darkroom conditions. The fluorescence spectrum data that excitation light central wavelengths were 370 and 395 nm were obtained. In order to accurately identify the useful information of fluorescence spectrum, the required fluorescence spectrum data segments (450~700 nm) were intercepted, and then processed them by removing background noise, normalization, wavelet transform, and so on. The above methods could amplify and identify the non-obvious fluorescence peaks in the fluorescence spectrum. The experimental results show that the fluorescence spectrum of corneal is detected within 420~600 nm when the LEDs with wavelengths of 370 and 395 nm are used as excitation sources. Moreover, the fluorescence spectra have peaks in the range of 450~500, 500~550 and 550~600 nm, respectively. According to the principle that the fluorescence peak of fluorescent substances is independent of the excitation wavelength, it is shown that the fluorescence spectra of two different excitation wavelengths are all produced by AGEs. The peak fluorescence intensity of diabetes mellitus patients and normal people were analyzed. The results showed that the fluorescence intensity of diabetes mellitus patients was significantly higher than that of normal people, which indicated that it was feasible to detect advanced glycation end products of the cornea by fluorescence spectroscopy.