Abstract: The unsaturated zone is the critical zone of the earth, which is closely related to the human living environment and health safety. Carbon dioxide plays a crucial role in the ecological environment and global climate change. In order to explore the potential risk of high concentration CO₂ invasion into the unsaturated zone to the ecological environment, and based on CO₂ simulation platform of in situ research filed of water and environment of the Chang’an University, we present a controlled experiment, in which 5%, 10% and 15% pure CO₂ was respectively injected into the local soil planted with five typical plants (ryegrass, wheat, corn, pea and amaranth) to assess the potential impact of CO₂ invasion on the environment, specifically on soil and plants. Botanical investigation and chemical analyses of soil samples (at depth from 20 to 30 cm) shows a significant adverse impact of CO₂ injection on plant growth, including declining plant height, number of leaves and fruit weight. Small changes were observed in mineralogy and bulk chemistry, showing the change of soil pH and reduction of nitrogen, available potassium, available phosphorus, etc. Different plants show different and complicated responses to CO₂ stress, depending on plant life history, including photosynthetic pathways, osmoregulation and antioxidant systems. On the whole, C3 monocotyledon ryegrass and C4 monocotyledon corn show greater sensitivity than other plants, suggesting their potential as indicator plants for evaluation of CO₂ impact on ecological environment.