A TFT substrate and a manufacturing method thereof are provided. In the manufacturing method, a metal oxide semiconductor layer is irradiated with UV light by using a gate as a shielding layer, such that a portion of the metal oxide semiconductor layer irradiated by the UV light is conductorized to form a source, a drain, and a pixel electrode, and a portion of the metal oxide semiconductor layer shielded by the gate still retains semiconductor properties to form a semiconductor channel. The invention achieves the alignment of the source and the drain with the gate by processes of self-alignment of the gate and conductorization of the metal oxide semiconductor layer, and can effectively control an overlapping region of the source and drain and the gate. Thereby, the parasitic capacitance is reduced, and the display quality is improved. Also, the manufacturing method is simple, and the production efficiency is improved.