Provided are a display substrate, a preparation method thereof, and a display apparatus. The display substrate includes a flexible substrate, a driving structure layer disposed on the flexible substrate, a light-emitting element disposed on the driving structure layer, and an encapsulation layer disposed on the light-emitting element. The flexible substrate includes a first flexible substrate layer, a first connection layer disposed on the first flexible substrate layer, and a second flexible substrate layer disposed on the first connection layer, wherein, the first connection layer includes at least one first via, and the first flexible substrate layer includes at least one first groove provided corresponding to the first via; the first via and the first groove are mutually communicated, and an orthographic projection of the first groove on the first flexible substrate layer includes an orthographic projection of the first via on the first flexible substrate layer.