A display panel is provided. The display panel includes a substrate, a light-shielding positioning layer and a transparent positioning layer. The substrate has a first surface and a second surface opposite to the first surface. The light-shielding positioning layer is disposed on the first layer and has at least one first alignment pattern. The transparent positioning layer is disposed on the second layer and has at least one second alignment pattern. In a direction perpendicular to the substrate, the at least one first alignment pattern overlaps with the at least one second alignment pattern. A manufacturing method of the display panel is also provided.