An OLED display substrate, a manufacturing method and a display device are provided. The OLED display substrate includes a base substrate and a plurality of pixel units arranged on the base substrate, each pixel unit includes a plurality of subpixel units, and each subpixel unit includes a switching TFT and a bottom-emission OLED, the OLED display substrate further includes a light-shielding layer arranged between the OLED and the switching TFT, and an orthogonal projection of the light-shielding layer onto the base substrate completely covers an orthogonal projection of a semiconductor region of the switching TFT onto the base substrate.