A light-emitting diode chip, a display substrate and a manufacturing method thereof are disclosed. The light-emitting diode chip includes a first conductive type semiconductor layer, a light-emitting layer, at least two second conductive type semiconductor layers, and at least two first electrodes; the at least two second conductive type semiconductor layers are at a side of the light-emitting layer away from the first conductive type semiconductor layer, the at least two first electrodes are electrically respectively connected with the at least two second conductive type semiconductor layers. Orthographic projections of the at least two second conductive type semiconductor layers on the first conductive type semiconductor layer are spaced apart from each other, and orthographic projections of the at least two first electrodes on the first conductive type semiconductor layer are spaced apart from each other.