A display substrate includes a first substrate, a first sub-pixel layer and a second sub-pixel layer positioned on the first substrate; the first sub-pixel layer includes a plurality of first sub-pixels, the second sub-pixel layer includes a plurality of second sub-pixels, and a first orthographic projection of the first sub-pixel on the first substrate does not overlap with a second orthographic projection of the second sub-pixel on the first substrate. A display panel, a display device and a method for manufacturing the display substrate are also disclosed.