This paper proposes a color separation algorithm for glass printing. The algorithm separates the original image into six kinds of color layers and can be easily applied to n kinds of arbitrary colors’ case. Given six kinds of ink, this algorithm mixes some of them up averagely to reach a variety of basic colors. The transparency channel is considered in the separation process, and any part of the image can be adjusted separately by setting its alpha value. After dealing with the transparency channel, color is separated by dithering method and the corresponding color layers are output at the same time. Moreover, the whole color separation process does not need any pre-treatment or post-treatment to do. Experiment shows that the original image can be appropriately restored on glass by using this algorithm and the transparency of the original image is well-expressed.