Embodiments of the present application provide an array substrate and a manufacturing method thereof, and a display panel. The array substrate includes a substrate, a source electrode, a drain electrode, a semiconductor layer, and a channel defining layer. The semiconductor layer is disposed between the source electrode and the drain electrode, and the semiconductor layer partially overlaps with the source electrode and the drain electrode. The channel defining layer is disposed around the semiconductor layer, and is correspondingly disposed on the source electrode and the drain electrode. When the semiconductor layer is prepared, the semiconductor layer is directly printed in a printing opening defined in the channel defining layer. Overflow of the semiconductor layer is prevented by the channel defining layer.