Provided are a display substrate and a preparation method thereof, and a display apparatus. The display substrate includes a display area, a mounting hole located in the display area, and an encapsulation region located between the display area and the mounting hole. The encapsulation region includes an insulating layer, an encapsulation pixel definition layer arranged on the insulating layer, and a partition structure layer arranged on the encapsulation pixel definition layer. The encapsulation pixel definition layer includes multiple spaced protrusions, the partition structure layer includes multiple spaced columns, the columns are arranged on the protrusions, a functional layer is arranged on the multiple spaced columns, and the functional layer is broken by the columns.