The invention provides a display substrate, a preparation method thereof, and a display device. The display substrate comprises a display area and a non-display area. The non-display area comprises afunctional layer, a retaining wall arranged on the functional layer, and an inorganic packaging layer covering the functional layer and the retaining wall. Between the display area and the retaining wall, the functional layer comprises a plurality of grooves arranged at intervals, so that a concave-convex structure is formed on the contact surface of the functional layer and the inorganic packaging layer. According to the invention, the plurality of grooves are formed in the area between the display area and the retaining wall at intervals, so a concave-convex structure is formed on the contact surface of the functional layer and the inorganic packaging layer, thereby increasing the bonding force of the film layer in the area, avoiding the delamination or cracking of an area between the display area and the retaining wall, avoiding a water and oxygen invasion path formed by delamination or cracking, and effectively guaranteeing the effectiveness and reliability of packaging. Moreover,the display substrate is low in manufacturing cost, is easy in process implementation and the like.