The molecular structure of glucose was explored. The diffuse reflectance Mid-Infrared (MIR) spectroscopy experiments were conducted. The infrared absorption modes of the main functional groups of glucose include free O-H functional group stretching vibration mode (ν■), dimeric O-H functional group stretching vibration mode (ν■), polymer O-H functional group stretching vibration mode (ν■), CH2 functional group asymmetric stretching vibration mode (ν■), C-H functional group stretching vibration mode (νC-H), CH2 functional group symmetric stretching vibration mode (ν■), water molecule O-H functional group bending vibration mode (δ■), coupled with of CH2 functional group bending vibration and several bending vibration of functional groups such as OCH, CCH, COH etc (δ■ + δ■ + δ■ +δ■), C-O functional groups in COH structure stretching vibration mode (ν■), C-O functional group on sugar ring structure stretching vibration mode (ν■), OCH functional group bending vibration (δOCH), C-O-H functional group bending vibration (δC-O-H), sugar ring structure asymmetric stretching vibration mode (ν■), C-C-H functional group and O-C-H functional group bending vibration (δC-C-H + δO-C-H), O-C-H functional group with α-terminal isomerism structure bending vibration (δO-C-H-α-terminal isomerism structure), sugar ring structure symmetric stretching vibration mode (ν■), functional group vibration mode caused by νC-C and νC-O. Diffuse reflectance MIR spectroscopy can rapidly investigate the molecular structure of glucose