In a method of manufacturing a thin film transistor substrate, a semiconductor pattern is formed on a substrate, a first etch stop layer and a second etch stop layer are sequentially formed on the semiconductor pattern, and the second etch stop layer and the first etch stop layer are sequentially patterned to form a second etch stop pattern and a first etch stop pattern. Thus, when the second etch stop layer is patterned using an etchant, the first etch stop layer covers the semiconductor pattern, thereby preventing the semiconductor pattern from being etched by the etchant.