The present disclosure relates to the field of display technology, and discloses a display substrate, a preparation method thereof, and a display panel, used for improving the structure of the display substrate, improving characteristics of a thin film transistor, and increasing the yield of a display product. The display substrate includes: a base substrate; a thin film transistor on the base substrate; and a flattening layer on a side, away from the base substrate, of the thin film transistor, wherein the flattening layer is provided with a groove around a channel area of the thin film transistor, and an orthographic projection of the groove on the base substrate is not overlapped with an orthographic projection of the channel area on the base substrate.