An organic light-emitting diode (OLED) display substrate, a manufacturing method thereof and a display panel are provided. The OLED display substrate has pixel regions and includes a base substrate and a pixel defining layer disposed on the base substrate; in regions of the pixel defining layer corresponding to the pixel regions, accommodation parts penetrating the pixel defining layer are disposed, and the pixel defining layer is further provided with guide parts disposed corresponding to the accommodation parts, the guide parts are located on a periphery of the corresponding accommodation parts and formed by recessed areas which are formed on a side of the pixel defining layer away from the base substrate, the recessed areas do not penetrate the pixel defining layer, and an orthographic projection of the guide part on the base substrate is directly coupled to an orthographic projection of the corresponding accommodation part on the base substrate.