A display substrate, a manufacturing method thereof, and a display panel are disclosed. The display substrate includes a glass substrate; plural sub-pixels; and plural light extraction modules disposed on the glass substrate at plural regions in one-to-one correspondence with the plural sub-pixels; wherein each of the light extraction modules has a hemisphere-shaped groove structure. By manufacturing the plural light extraction modules each having a hemisphere structure and a higher refractive index on the glass substrate in one-to-one correspondence with the plural sub-pixels, the refraction angle of the light that is emitted from each of the sub-pixels and incident onto the glass substrate can be decreased, so that more light can be emitted out; in this way, it is possible to improve the luminous efficiency of the display substrate and hence the display effect of the display device.