Near-infrared spectroscopy (NIRS) is a powerful tool for the rapid identification and quantitative analysis of dairy farm slurry components. Temperature variations significantly influence on the spectra measurements while their underlying spectral response mechanisms remain insufficiently understood. This study investigates the impact of temperature variations on the spectral characteristics of slurry to ascertain the availability of improving the NIRS-based analysis. Using a portable detection device of slurry nutrients, near-infrared diffuse reflectance spectra were acquired from slurry samples across temperatures ranging from 0 to 40 °C. Principal component analysis (PCA) and two-dimensional correlation spectroscopy (2D-COS) were applied to assess spectral response intensity, temporal sequence and correlations among functional groups. The results indicate that increasing temperature weakens hydrogen bonding strength, leading to intensity changes. In particular, hydrogen-bonded water partially dissociates, enhancing features associated with free O[sbnd]H groups and indicating temperature-induced changes in intermolecular interactions. Additionally, elevated temperatures increase the susceptibility of specific absorption bands ((N[sbnd]H), and (O[sbnd]H)) to shift, affecting the stability of slurry composition predictions. 2D-COS analysis reveals the sequential evolution of absorption band changes of specific functional groups under thermal perturbation, offering insight into dynamic transformations. While 2D-COS has been previously applied to various agricultural materials, this study extends its application to temperature-induced spectral analysis of dairy farm slurry, addressing a specific challenge in NIRS-based nutrient analysis where conventional temperature correction approaches have shown limitations. By identifying temperature-sensitive spectral regions and molecular transformations, this study provides a scientific basis for developing temperature-resilient NIRS models, which are crucial for facilitating real-time nutrient monitoring and precision slurry management on dairy farms