The diagnosis of diffuse axonal injury (DAI) is an important task in forensic pathology and clinical medicine. This study aimed to explore the use of Fourier transform infrared spectroscopy (FTIR) to detect DAI. The DAI area of the rat model was detected point by point by the FTIR-mapping system. Infrared spectral data of DAI were obtained by selecting the amide A band, CH3 symmetric stretching, collagen triple-helix structure and asymmetric stretching vibrational frequency of nucleic acid and phospholipid PO2 as the target peak positions. The system can automatically draw infrared spectral color pathological images. In the DAI group, the amide A protein secondary amine N-H stretching vibration and the collagen triple-helix structure of the high-absorption area were consistent with the DAI area confirmed by the silver and β-APP staining. The CH3 symmetric stretching, nucleic acid and phospholipid PO2 symmetric stretching vibration absorption spectra showed no significant differences between the experimental and verification groups. The FTIR-mapping technique can visually express the molecular characteristics of DAI, which is expected to be applied to the pathological diagnosis of DAI.