The use of Raman hyperspectral imaging technique can obtain not only the spatial distribution information of samples, but also the spectral information of each pixel on the image. It has been applied in food safety testing for its abundant information. This study was aimed at quantitatively analyzing of light penetration depth through milk powder in Raman hyperspectral imaging, and exploring the influences of system parameters and types of milk powders. The attenuation of Raman signal in milk powder layer was also discussed. Milk powder layers with five different thicknesses ranging from 0.8~4.0 mm were prepared on the top of a melamine layer with 5 mm thick. The penetration depth of light was evaluated by detecting the characteristic peaks of melamine. The results showed that the Raman characteristic signal was enhanced with a bigger laser intensity and longer exposure time at the same thickness of milk powder layer. When the laser intensity was not less than 2 W and the exposure time was not less than 500 ms, the light penetration depth in whole milk powder was 4 mm at least. The peak intensities of melamine decayed in exponent with the thickness of milk powder layer increasing from 0.8 to 4.0 mm. The light penetration depths in whole milk powder, low fat milk powder and skim milk powder were all 4 mm at least when the laser intensity was 8 W and the exposure time was 1 000 ms. The Raman signal through the skim milk powder layer was weaker than those through the whole milk powder layer and the low fat milk powder layer at the same thickness. Therefore, the light penetration depth of the skim milk powder layer was less than those of the whole milk powder layer and the low fat milk powder layer. The results provide a useful reference for the pretreatment of milk powder in safety and quality detection by Raman hyperspectral imaging.