The invention provides an array substrate and a preparation method thereof, a display panel and a display device. The array substrate comprises a substrate body and a flat layer, an anode, a light-emitting function layer, a cathode and a pixel defining layer, which are arranged on the substrate body. The pixel defining layer is used for defining a pixel opening area; the light-emitting functionallayer is arranged in the pixel opening area; the flat layer, the anode, the light-emitting functional layer and the cathode are sequentially arranged away from the substrate; and the pixel defining layer is located between the flat layer and the cathode. The array substrate also comprises a middle insulating layer, wherein the middle insulating layer is arranged between the flat layer and the pixel defining layer, and the chemical polarity of the middle insulating layer is between the chemical polarity of the flat layer and the chemical polarity of the pixel defining layer. According to the array substrate, by arranging the middle insulating layer, the chemical polarity difference between the pixel defining layer and the flat layer is greatly reduced, the pixel defining layer and the flatlayer can be more firmly adhered together through the middle insulating layer, so that the pixel defining layer is prevented from falling off, and the quality of the pixel defining layer is ensured.