A display substrate, a manufacturing method thereof, and a display device are provided. A first test signal line in a test circuit in the display substrate extends in a second direction, and a second test signal line includes a first part extending a the first direction and a second part extending in the second direction; a width of the second part of the second test signal line in a direction perpendicular to the second direction is different from a width of the first test signal line in the direction perpendicular to the second direction; a difference between a resistance of the first test signal line and a resistance of the second test signal line the difference is less than a first threshold. The display substrate as provided is used for display.