A manufacturing method of a color filter substrate is provided. The manufacturing method includes steps of providing a substrate comprising a color filter region and a black matrix region; depositing a black matrix layer on a surface of the substrate in the black matrix region; applying an RGB color resist to the color filter region of the substrate; correspondingly forming a plurality of photo spacers on a surface of the black matrix, the photo spacers being made from light-shielding material; forming a protective layer above the substrate. The photo spacers made from light-shielding material is arranged on the surface of the black matrix to prevent the transmission of light rays through the RGB color resist that results in mixture of colors.