A quantum dot light-emitting device, a fabricating method thereof, and a display substrate are provided. The quantum dot light-emitting device includes: a base substrate; a first electrode layer, a light-emitting layer, a second electrode layer and an encapsulation layer which are sequentially formed on the base substrate, wherein the light-emitting layer includes a quantum dot light emitting material; a fluorescent material is disposed between the first electrode layer and the second electrode layer, and the fluorescent material includes a thermally activated delayed fluorescence (TADF) material; one of the first electrode layer and the second electrode layer is an anode layer, and the other of the first electrode layer and the second electrode layer is a cathode layer.