A display substrate motherboard has display areas and at least one cutting area located at least among the display areas. The display substrate motherboard includes a base substrate, first barrier groups disposed on the base substrate, an inert material layer and a first inorganic insulating layer. Each first barrier group is located on at least one side of a corresponding display area and includes two first barriers. A region between the two first barriers is located in a cutting area. The inert material layer is disposed in the region. The first inorganic insulating layer is disposed on a side of the first barrier groups away from the base substrate, and is further located in the display areas. An orthogonal projection of the first inorganic insulating layer on the base substrate is non-overlapping with an orthogonal projection of the inert material layer on the base substrate.