The near-infrared absorption features are easily affected by temperature changes, which reduce the accuracy of quantitative analysis. This paper presents the methods for analysing the source and amplitude of temperature disturbance based on the theory of two-trace two-dimensional correlation spectroscopy (2T2D-COS). The absorption spectra of glucose solution, with a concentration of 0～200 mg·dL-1, are measured in the temperature range of 34~38.5 ℃ with a stepwise of 0.5 ℃. After baseline correction and noise filtering, the concentration-dependent and temperature-dependent spectra are analyzed with the 2T2D-COS algorithm. In the asynchronous spectrum of temperature-dependent spectra, the cross-peaks are observed at 1 474 and 1 410 nm, corresponding to strong and weak hydrogen-bonded water, respectively. While in the asynchronous spectrum of concentration-dependent spectra, the cross peaks at 1 450 and 1 595 nm are related to the characteristic absorption of water and glucose. From the slice spectrum at 1 410 nm, the intensity of the peaks in the range of 1 410n~1 600 nm is increased with temperature, demonstrating good correlation between the cross peaks and temperature variance. The calibration model is established for the quantitative analysis of sample temperature with the cross peak intensity range of (1 475±4) nm. The root mean square error achieved 0.125 9 ℃ for sample temperature prediction with linear regression. The experimental results demonstrate that the temperature disturbances could be identified and qualified with distinctly separate cross peaks in the asynchronous spectrum, obtained using only a pair of spectra. The proposed approach also provides a reasonable way for researchers to simplify the in vivo measurement system and establish a more stable calibration model.