The invention discloses a display substrate, a manufacturing method thereof and a display device, and belongs to the field of display. The display substrate comprises a display region and a frame adhesive applying region. The frame adhesive applying region is located outside the display region. A flow limiting organized array is arranged at the edge of the side, away from the display region, of the frame adhesive applying region. The flow limiting organized array comprises at least two adhesive containing grooves, and the adhesive containing grooves are formed in the direction away from the display region. Outward-overflowing frame adhesives are made to sequentially flow into the multiple adhesive containing grooves formed from interior to exterior, the flow limiting organized array can limit or block the frame adhesive from overflowing outwards, and the outer side structure is prevented from being covered with the frame adhesives, so that the influence caused by overflowing of the frame adhesives on the follow-up process is reduced, and the yield is improved.