A thin film transistor substrate with good process efficiency and a method of manufacturing the same are provided. The thin film transistor substrate includes a first conductive type MOS transistor and a second conductive type MOS transistor. The first conductive type MOS transistor includes a first semiconductor layer formed on a blocking layer and having first conductive type low-concentration doping regions adjacent to both sides of a channel region, first conductive type source/drain regions adjacent to the first conductive type low-concentration doping regions, a first gate insulating layer formed on the first semiconductor layer, a second gate insulating layer formed on the first gate insulating layer and overlapping with the channel region and the low-concentration doping regions of the first semiconductor layer, and a first gate electrode formed on the second gate insulating layer. The second conductive type MOS transistor includes a second semiconductor layer formed on the blocking layer and having second conductive type source/drain regions adjacent to both sides of a channel region, the first gate insulating layer formed on the second semiconductor layer, a third gate insulating layer formed on the first gate insulating layer and overlapping with the second semiconductor layer, and a second gate electrode formed on the third gate insulating layer.