The molecular structure and stability of glucose were explored. The diffuse reflectance and in-situ diffuse reflectance mid infrared (MIR) spectroscopy experiments and statistical calculations of related spectral data were conducted. The results showed that the infrared absorption modes of the main functional groups of glucose include: polymer O-H functional group stretching vibration mode (ν■), asymmetric stretching vibration mode of CH2 functional group (ν■), stretching vibration mode of C-H functional group (νC-H), symmetric stretching vibration mode of CH2 functional group (ν■), stretching vibration mode of C-O functional group in COH structure (νC-O-COH), deformation vibration mode of OCH functional group (δOCH), deformation vibration mode of C-O-H functional group (δC-O-H), bending vibration of CH2 functional group and coupling of several angle changing vibrations of OCH, CCH, COH and other functional groups (δ■ + δOCH + δCCH + δCOH), coupling mode of angle changing vibration of C-C-H functional group and O-C-H functional group (δC-C-H + δO-C-H), vibration coupling modes of functional groups induced by νC-C and νC-O (νC-C + νC-O), asymmetric stretching vibration mode of sugar ring structure (ν■), stretching vibration mode of C-O functional group in sugar ring structure (ν■), the angular vibration mode of O-C-H functional group with α-end group isomerism (δ■), the symmetric stretching vibration mode of sugar ring structure (ν■) and the vibration mode caused by skeleton vibration (ν-skeleton). The stability of the hydroxyl structure, sugar ring structure, and skeleton structure corresponding to the ν■, ν■, and ν■,ν■ and ν-skeleton-2 is poor. The CH2 structure corresponding to ν■ and the hydroxyl structure corresponding to ν■ are relatively stable. Diffuse reflectance and in situ diffuse reflectance MIR spectroscopy can rapidly investigate the molecular structure of glucose.