The effect of temperature on the fluorescence of milk powder was studied by fluorescence spectra and fluorescence lifetime technology. Firstly, the fluorescence of milk powder is mainly from casein determined by comparing the fluorescence spectra of milk powder, casein, lactose and vitamins. Secondly, as the temperature increases, the fluorescence intensity of infant formula powder, toddler formula powder, adult ordinary milk powder and casein are generally reduced. The trend of fluorescence intensity with temperature of toddler formula powder and adult ordinary milk is almost same. The change of the fluorescence intensity of infant formula powder with temperature is different, which shows a relatively slow-changing platform area. Casein′s fluorescence strength is a slow downward with increasing temperature, falling slower than milk. When heated to 90 degrees and then cooled to 25 degrees, casein was partially recovered, indicating that changes in the activity of the protein structural or fluorophore are partially reversible. Finally, the fluorescence lifetime of milk powder and casein at different temperatures were measured. Milk powder and casein have two lifetimes after double exponential fit, which can be derived from tryptophan and phenylalanine. As the temperature increases, fluorescence lifetimes of milk powder and casein are decreasing.