A display substrate including a drive-circuit layer and a light-emitting structure layer, a preparation method thereof, and a display device, the light-emitting structure layer includes an anode, a pixel definition layer, an organic light-emitting layer and a cathode, and an auxiliary electrode and an organic light-emitting block, arranged sequentially, the pixel definition layer includes an anode opening exposing the anode and an electrode opening exposing the auxiliary electrode, the organic light-emitting block is separated from the organic light-emitting layer, the auxiliary electrode includes the first, second and third auxiliary electrodes arranged sequentially; the cathode includes a first horizontal lapping part lapping with the first auxiliary electrode and a second sidewall lapping part lapping with the second auxiliary electrode, the thickness of the second sidewall lapping part in the direction parallel to the substrate is greater than that of the first horizontal lapping part in the direction perpendicular to the substrate.