Tunnel construction undermines groundwater circulation system of karst areas and leads to groundwater level drop, which might exert influence on surrounding ecological environment. Taking karst valley area of Zhongliang Mountains in Chongqing as an example, we conducted a comparative analysis on soil water content at different depths soil layers (0-20 cm and 20-40 cm) and Instantaneous water use efficiency (WUE_inst) of typical plants(evergreen arbor and evergreen shrub) in areas affected and unaffected by tunnel in both dry and rainy seasons to explore responses of plants WUE_inst to tunneling works. The results showed that evaporation and precipitation mainly led to similarity of soil water content of different depths and in different seasons at both areas. However, soil water content of areas affected by tunnel was significantly higher than that of areas unaffected by tunnel in both dry and rainy seasons. The WUE_inst of plants varied greatly with seasons at both areas and the WUE_inst of various plants in dry season was significantly higher than that in rainy season. The WUE_inst of plants of areas affected by tunnel was significantly higher than that of areas unaffected by tunnel in both dry and rainy seasons. Additionally, the results of correlation analysis between WUE_inst of plants and soil water content showed that there was a negative correlation between the WUE_inst of plants and soil water content. In comparison with areas unaffected by tunnel, the WUE_inst of plants in areas affected by tunnel was more sensitive to changes of soil water content. The above results suggested that the tunneling works led to loss of groundwater resources, decrease of soil water content, and change of WUE_inst of plants. Consequently, it is required to take more conservative water use strategies in response to certain water stress on plants in areas affected by tunnel construction.