An organic light-emitting diode and a preparation method thereof, a display substrate, and a display device. The organic light-emitting diode includes a double-layer fold structure, the double-layer fold structure includes a first layer and a second layer adjacent to each other, an interface between the first layer and the second layer and a surface of the second layer farther away from the first layer have a fold morphology, a virtrification transition temperature of the first layer is less than a virtrification transition temperature of the second layer, and a thermal expansion coefficient of the first layer is greater than a thermal expansion coefficient of the second layer. In the organic light-emitting diode, a natural and self-assembled fold morphology can be obtained with a double-layer thin film, to replace an external fold structure, and the resulted device has a higher efficiency and a reduced operating voltage.