When plants are continuously treated with high concentration of CO2, they will show different physiological and biochemical characteristics due to the different conditions of the plants themselves and the environment, and the leaves, as the main organ of gas exchange between plants and the outside world, will first respond to CO2 stress through photosynthesis and respiration when the atmospheric concentration of CO2 rises. In this study, tetraploid Sophora japonica was selected as the research object to analyze the stomatal movement pattern and the changes of photosynthesis and respiration under high CO2 culture conditions, and we found that ROS and NO were synthesized and participated in the regulation of CO2-mediated stomatal closure, and we also proved that the alternate respiration pathway could alleviate the oxidative damage caused by high CO2 concentration. Combined with the proteomics approach, the key proteins responsive to high concentration of CO2 were mined according to the changes of differential proteins under different treatment conditions, with the aim of laying a theoretical foundation for the study of elucidating the response mechanism of tetraploid Sophora japonica under high concentration of CO2 treatment.