A method for manufacturing spacers and a method for manufacturing a display substrate are disclosed. The method for manufacturing spacers includes performing an over-development process on the photoresist layer processed by the exposure process to form a first photoresist pattern and a second photoresist pattern on the substrate; an outer edge of a lower portion of the first photoresist pattern near the substrate is provided with a recess, and an outer edge of a lower portion of the second photoresist pattern near the substrate is provided with a recess; waiting a first duration for photoresist on a surface of the first photoresist pattern to flow downwards to fill the recess of the first photoresist pattern, and photoresist on a surface of the second photoresist pattern to flow downwards to fill the recess of the second photoresist pattern.