The invention relates to an OLED display substrate comprising a substrate, a thin film transistor array layer on the substrate, a flat layer, an anode on the flat layer, a pixel defining layer, a cathode and a light-emitting layer, wherein a pixel region is defined by the pixel defining layer, the light-emitting layer is in the pixel region, and the OLED display substrate also comprises a light guiding layer which is disposed in contact with the light-emitting layer and is used for guiding light of the light-emitting layer out. The invention also relates to an OLED display device and a brightness compensation method. By setting the light guiding layer, optical parameters are collected from the inside of the display substrate, therefore, the optical compensation is carried out, the uniformity of brightness after compensation is improved, and the structure is simple.