Provided are a display substrate and a preparation method thereof, and a display device. The display substrate includes a base substrate, a light-emitting element disposed on the base substrate, and an encapsulation layer disposed on the light-emitting element, the display substrate further comprises a driving circuit connected with the light-emitting element and configured to drive the light-emitting element, the driving circuit comprises a driving transistor which comprises an active layer located inside the base substrate; the encapsulation layer comprises a first gradient layer and a second gradient layer stacked along a direction away from the base substrate, a content of oxygen element in the first gradient layer gradually decreases, and a content of carbon element in the second gradient layer gradually increases along the direction away from the base substrate.