A test substrate includes a base and a first electrode layer, a pixel defining layer, a light-emitting functional layer and a second electrode layer disposed on the base in sequence. The test substrate has at least two test regions, and each test region is a region where one first electrode of the plurality of first electrodes is located. Each test region includes a first region. Orthographic projections of portions of the pixel defining layer and the light-emitting functional layer located in a same first region on the base overlap with each other, and areas of orthographic projections of portions of the first electrode layer located in first regions of the at least two test regions are different.