The present disclosure discloses a quantum dot display substrate and a manufacturing method thereof, and a quantum dot display device. The quantum dot display substrate comprises black matrix patterns, quantum dot patterns surrounded by the quantum dot patterns, a first base substrate, and a first optical layer and a second optical layer both arranged on a side of the first base substrate, and the first optical layer is arranged on a side of the second optical layer away from the first base substrate; the first optical layer is configured to collimate incident light to generate collimated light and transmit the collimated light to the second optical layer; the second optical layer is configured to adjust an outgoing direction of the collimated light to generate outgoing light, and make the outgoing light irradiate onto the black matrix patterns.