Abstract: This article collects the co-seismic response changes of the temperature and water level of Jiangsu fluid wells during the M7.4 earthquake in Maduo, Qinghai, summarizes their characteristics, analyzes the influencing factors in combination with the basic parameters of Jiangsu’s underground fluid wells and regional geological structures, and explores the changing mechanisms. The results show that the impact of the M7.4 earthquake in Maduo, Qinghai on underground fluids in Jiangsu was relatively significant, and the recording ability of the water level’s co-seismic response was better than that of the temperature. The temperature mainly showed step changes, while the water level mainly showed oscillations and step changes. The fluid wells that exhibited co-seismic response changes were mainly concentrated near the Tanlu fault zone and Maoshan fault zone. The regional and concentrated step changes in water level indicate that stress adjustment has occurred in the area, which has some indicative significance for the future occurrence of earthquakes. The co-seismic response mechanism of water temperature is more complicated. The movement mode and flow rate of water, wellbore conditions, and probe position may all affect its changes. More data is needed from various aspects to make a more accurate judgment.