Three-dimensional fluorescence spectra of milk of three brands (9 types) were measured by FLS920P fluorescence spectrometer, including pure milk and fresh milk, whole milk and skim milk. The fluorescence peak wavelength of milk is about 349 nm, the full width at half maximum (FWHM) is about 66 nm, the optimal excitation wavelength is about 291 nm, and the average lifetime is about 4.6 ns, the results show that the fluorescence spectrum of 9 kinds of milk is basically the same except the fluorescence intensity. The fluorescence peak wavelength of the casein solution is 344 nm, the FWHM are 66 nm, the optimal excitation wavelength is 295 nm, and the fluorescence lifetime is 4.1 ns. The fluorescence peak wavelength and fluorescence lifetime of casein are similar to that of milk. After comparing other fluorescent substances in milk, it is inferred that casein is the main fluorescent substance in milk. In order to explore the differences in fluorescence intensity of 9 types of milk, the fluorescence emission spectra under the optimal excitation wavelength of 9 types of milk are compared. The fluorescence intensity of the whole milk of the same brand is significantly lower than that of skim milk. By comparing the normalization fluorescence spectra of whole milk, skim milk and centrifuged whole milk, the fluorescence intensity of centrifuged whole milk is between that of whole milk and that of skim milk. Centrifugation of milk results in less fat and less scattering and increase fluorescence intensity. The full fluorescence emission spectrum of milk shows that the Rayleigh scattering intensity of whole milk is significantly higher than that of skim milk, whole milk has a high-fat content and strong scattering, the excitation light is scattered more after it enters the whole milk, resulting in the Rayleigh scattering intensity of the whole milk being higher than that of skim milk. The light transmittance curve shows that the transmittance of whole milk is lower than that of skimmed milk, when incident light passes through whole milk, in addition to the part of it being absorbed by casein, part of it is lost due to fat scattering, and the transmittance is reduced, so that thetransmittance of whole milk is lower than that of skim milk. In this paper, the main fluorescent substances of milk are determined by using fluorescence spectroscopy without pretreatment. Moreover, the reason for the difference in fluorescence intensity between whole milk and skim milk is explained.