A color filter substrate, a display panel and a method of manufacturing color filter substrate are provided. The color filter substrate comprising a first glass substrate and a blocking wall formed on a surface of the first glass substrate which is toward to an array substrate, the blocking wall is a projection structure of the surface of the first glass substrate, and the blocking wall is located in a non-display region. The invention further provides correspondingly display panel and method of manufacturing color filter substrate. The color filter substrate, display panel and method of manufacturing color filter substrate of this disclosure, which using the glass substrate on CF side to be aligning film blocking wall. It could control boundary of the aligning film on CF side, and prevent water vapor pass into inside panel of the aligning film on CF side caused circuit corrosion.