The invention is suitable for the technical field of displays, and provides a chip on film (COF) substrate, which comprises a substrate, a protective layer and a metal film protective layer, wherein a welding wire is arranged on the surface of the substrate; the protective layer is arranged in the preset position of the welding wire; the metal film protective layer is arranged on the part, uncoated with the protective layer, of the welding wire. The active metal film protective layer is plated on the welding wire partially exposed in the air on the COF substrate, conduction is not affected, the time of copper corrosion is delayed, and the speed of copper corrosion is slowed. By applying the COF substrate and the manufacturing method thereof and the display panel, disclosed by the invention, the fracture risk of the welding wire on the COF substrate due to corrosion is effectively reduced, thus the using time of a product is increased, and the service life of the product is prolonged.