Provided is a method for preparing a display substrate. The display substrate includes multiple pixel island regions, empty regions and connection bridge regions. The preparation method includes: forming first grooves corresponding to the pixel island regions and second grooves corresponding to the connection bridge regions on a side of a hard underlay substrate; preparing the display substrate on a side of the underlay substrate where the first grooves and the second grooves are formed, wherein the pixel island regions are located in regions where the first grooves are located, the connection bridge regions are located in regions where the second grooves are located, and the empty regions are located in regions other than the first grooves and the second grooves; and separating the underlay substrate from the display substrate to obtain the display substrate.