The present disclosure provides a display substrate, a manufacturing method thereof and a three-dimensional display apparatus. The display substrate includes a base substrate with a plurality of sub-pixels. Each of the sub-pixels includes at least two first electrodes, and a light-emitting function layer disposed on a side of the first electrodes facing away from the base substrate. Each first electrode includes: a transparent conductive portion and a reflective conductive portion arranged in stack. In at least one of the sub-pixels, two adjacent first electrodes correspond to one reflective structure, the reflective structure includes a first portion and a second portion, an orthographic projection of the first portion on the base substrate and an orthographic projection of one first electrode have an overlap area, and an orthographic projection of the second portion on the base substrate an orthographic projection of another first electrode have an overlap area.