A light-emitting diode substrate and a manufacturing method thereof, and a display device are provided. The manufacturing method of a light-emitting diode (LED) substrate, including: disposing a supporting substrate supporting a plurality of LED units to be opposed to a receiving substrate so that a side of the supporting substrate facing the receiving substrate supports the plurality of LED units; and irradiating a side of the supporting substrate away from the receiving substrate with laser, stripping the LED units from the supporting substrate, and transferring the LED units onto the receiving substrate. The manufacturing method of the LED substrate can better transfer LED units from the supporting substrate onto the receiving substrate.