The invention discloses an electroluminescent device based display substrate, a preparation method thereof and a display device. The display substrate comprises a substrate body, the substrate body isprovided with an electroluminescent device, a pixel definition structure and a thin film transistor for controlling the electroluminescent device, the substrate body is further provided with a conducting layer connected with an electrode of the electroluminescent device, the conducting layer is overlapped with an active layer of the thin film transistor, and the overlapped area is covered with the pixel definition structure. Thus, the display substrate can realize electric connection between the electroluminescent device and the thin film transistor needless of a through hole etching or trepanning manner, the production technology can be simplified, the overlapped area has a higher transmittance by means of the overlapping manner, and the total transparent degree of the display substrateis improved; and an aperture ratio of the display substrate is large, the luminescent area is large, the luminescent efficiency is high, both the transmittance and luminescence efficiency can be takeninto consideration, and the product is more competitive in the market.