A display substrate includes pixel-defining layer and sub-pixels of N colors, wherein N≥3. The pixel-defining layer defines a plurality of sub-pixel areas having the sub-pixels arranged in therein; the pixel-defining layer includes a first pixel-defining layer and a second pixel-defining layer; a height of the second pixel-defining layer is smaller than a height of the first pixel-defining layer; and the first pixel-defining layer is configured to separate adjacent sub-pixels of different colors; the second pixel-defining layer is configured to separate adjacent sub-pixels of the same color; and the sub-pixels of N colors include red sub-pixels, blue sub-pixels, and green sub-pixels; the display substrate includes at least one column of sub-pixels, and in the at least one column of sub-pixels, sub-pixels belonging to a same column have a same color; and in a column of sub-pixels of the same color, corresponding light-emitting layers of at least two adjacent sub-pixels are connected.