The present disclosure provides a 3D display substrate, its manufacturing method and a 3D display device. The 3D display substrate includes a base substrate, a color filter layer arranged on the base substrate, and an optical structure layer arranged on the base substrate and at a light-exiting side of the color filter layer. The optical structure layer includes a first light-transmitting layer and a second light-transmitting layer having different refractive indices. An interface between the first light-transmitting layer and the second light-transmitting layer is of a concave-convex structure, so as to enable one of the first light-transmitting layer and the second light-transmitting layer to form a lens array structure for 3D display.