A method for manufacturing a display substrate comprises: forming a pixel definition layer on a lower surface of a base so as to define a pixel area; forming a spacer on a lower surface of the pixel definition layer; arranging a mask plate under the spacer; and moving an evaporation source along a first direction parallel to the lower surface of the base, so as to form an organic light emitting layer in the pixel area. When the evaporation source starts forming the organic light emitting layer in the pixel area, the spacer and the mask plate do not block a first border of the evaporation area. Further, when the evaporation source stops forming the organic light emitting layer in the pixel area, the spacer and the mask plate do not block a second border of the evaporation area.