A substrate and a preparation method thereof, a display panel and a preparation method thereof, and a display device are provided. The substrate includes a display region and a peripheral region positioned in a periphery of the display region and used for sealing, the substrate includes: a base substrate; an insulating layer, arranged on a side of the base substrate and positioned in the display region and the peripheral region for sealing; and a plurality of pixel units, positioned on the insulating layer corresponding to the display region, and in the peripheral region, at least one groove is disposed on a side of the insulating layer which faces away from the base substrate, a side of the groove which is away from the base substrate is open, and a depth direction of the groove is perpendicular to the base substrate.