Embodiments of the present invention provide a display substrate, a display device, a pressure detection system and a detection method thereof, which relate to the field of display technologies and is able to integrate the ambient pressure testing function in the display substrate for the convenience of being carried by people when going out, wherein the display substrate comprises a base substrate and a pressure sensing structure located on the base substrate configured to test the ambient pressure. The pressure sensing structure comprises a first pressure sensitive electrode and a second pressure sensitive electrode arranged opposite to each other, and a plurality of insulating pillars arranged at intervals between the first pressure sensitive electrode and the second pressure sensitive electrode, wherein the first pressure sensitive electrode is in contact with the base substrate.