A display panel and a manufacturing method thereof, and an alignment method are provided. The display panel includes a display substrate and a cover plate connected through an optical adhesive layer. The display substrate includes a plurality of light emitting elements, a pixel defining layer, and an encapsulation layer; the encapsulation layer includes a first inorganic encapsulation layer, a first organic encapsulation layer, and a second inorganic encapsulation layer; the display panel includes a display region and a frame region including a cutting region and a dam glue region where a dam glue is located; at least one of the plurality of cover plate alignment marks is between the dam glue and the display region and is overlapped with the second inorganic encapsulation layer, and at least one of the plurality of cover plate alignment marks is between the dam glue and the cutting region.