Three-dimensional fluorescence spectra of pure milk, milk with high calcium, milk with high calcium and low fat, and pure milk expiring by about 10 days are researched. The experiment indicates that the fluorescence peak of milk aqueous solution shows red shift with the increase of stimulation wavelength. When the stimulation wavelength is longer than 340 nm, a secondary fluorescence peak appears in the fluorescence spectrum, and it also shows red shift with the increase of stimulation wavelength. By comparison it is found that the concentration has little effect on fluorescence spectrum of milk aqueous solution, while the species of milk has greater impact, especially the milk with high calcium and low fat. The reason why the fluorescence intensities of the milk with high calcium and low fat and the expired milk are stronger under certain stimulation wavelength is analyzed preliminary. The findings may provide reference when using the fluorescence spectrum to identify milk quality.