A display substrate and a preparation method thereof, and a display device are provided. The display substrate (200/300/400) includes a display region (201/301/401) and an opening region (2011/3011/4011), the display region (200/300/400) surrounds the opening region (2011/3011/4011), a first barrier wall (2012/3012/4012) is between the display region (201/301/401) and the opening region (2011/3011/4011), and the first barrier wall (2012/3012/4012) surrounds the opening region (2011/3011/4011); the first barrier wall (2012/3012/4012) includes a first metal layer structure, and at least one side surface, surrounding the opening region (2011/3011/4011), of the first metal layer structure includes a recess (2012A/3012A/4012A). The display substrate combines the imaging device with the display region of the display substrate and has a better encapsulation effect.