A display substrate and a manufacturing method thereof. The display substrate includes a display region, a barrier region and an opening region, the barrier region is located between the display region and the opening region. The barrier region includes a first barrier wall, a first barrier wall, and a second barrier wall which are sequentially arranged from the display region to the opening region. The first barrier wall includes a first metal layer structure, at least one lateral surface of the first metal layer structure surrounding the opening region is provided with a notch; the first intercepting wall includes a first insulating layer structure; the second barrier wall includes a second metal layer structure and a first stacked structure, at least one lateral surface of the second metal layer structure surrounding the opening region is provided with a notch.