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To non-destructively and rapidly monitor the chlorophyll content of winter wheat leaves under CO₂ microleakage stress, and to establish the quantitative relationship between chlorophyll content and sensitive bands in the winter wheat growing season from 2023 to 2024, the leakage rate was set to 1 L/min, 3 L/min, 5 L/min, and 0 L/min through field experiments. The dimensional reduction was realized, fractional differential processing of a wheat canopy spectrum was carried out, a multiple linear regression (MLR) and partial least squares regression (PLSR) estimation model was constructed using a SPA selection band, and the model’s accuracy was evaluated. The optimal model for hyperspectral estimation of wheat SPAD under CO₂ microleakage stress was screened. The results show that the spectral curves of winter wheat leaves under CO₂ microleakage stress showed a “red shift” of the green peak and a “blue shift” of the red edge. Compared with 1 L/min and 3 L/min, wheat leaves were more affected by CO₂ at 5 L/min. Evaluation of the accuracy of the MLR and PLSR models shows that the MLR model is better, where the MLR estimation model based on 1.1, 1.8, 0.4, and 1.7 differential SPAD is the best for leakage rates of 1 L/min, 3 L/min, 5 L/min, and 0 L/min, with validation set R² of 0.832, 0.760, 0.928, and 0.773, which are 11.528, 14.2, 17.048, and 37.3% higher than the raw spectra, respectively. This method can be used to estimate the chlorophyll content of winter wheat leaves under CO₂ trace-leakage stress and to dynamically monitor CO₂ trace-leakage stress in crops.