The invention relates to an organic light-emitting display substrate, a preparation method thereof and an organic light-emitting display device. The organic light-emitting display substrate comprisesa display area and an opening area located in the display area. The preparation method of the organic light-emitting display substrate comprises the steps: forming a flexible substrate on a supportingsubstrate, wherein the flexible substrate comprises an open pore, and the open pore is located in the open pore area; forming an organic light-emitting device layer on one side, far away from the support substrate, of the flexible substrate; forming a packaging layer on one side, far away from the flexible substrate, of the organic light-emitting device layer, wherein the packaging layer covers the organic light-emitting device layer and the opening, the packaging layer comprises a first to-be-etched part, the first to-be-etched part is located in the open pore, and the extending direction ofthe first to-be-etched part intersects with the side wall of the open pore; etching the first to-be-etched part, and removing the first to-be-etched part; and stripping the support substrate from theflexible substrate to obtain the organic light-emitting display substrate. According to the embodiment of the invention, the defect that the packaging layer is peeled off due to the fact that the supporting substrate is peeled off from the flexible substrate can be overcome.