An organic thin film transistor substrate and a method of manufacturing the organic thin film transistor substrate capable of preventing overflow of an organic semiconductor layer. An organic thin film transistor substrate comprises a gate line formed on the substrate, a data line intersecting the gate line, a thin film transistor connected to the gate line and the data line and including an organic semiconductor layer, a pixel electrode connected to the thin film transistor, an organic protective layer protecting the thin film transistor, a first bank-insulating layer providing filling areas in the organic gate insulating layer and the organic semiconductor layer, and a second bank-insulating layer providing the filling area of the organic semiconductor layer together with the first bank-insulating layer and formed on a source electrode and a drain electrode of the thin film transistor.