Aloe is widely used in various fields for its rich polysaccharides, proteins, amino acids, vitamins, active enzymes and trace beneficial elements to human body. However, the main active ingredient aloin is also an allergenic ingredient, which even may cause a severe allergic reaction. In this study, infrared spectroscopy, Raman spectroscopy applied to the structural characterization of the aloin. Density functional theory (DFT) is applied to the theoretical calculations using the B3LYP/6-31G (d) basis set vibration, which was helpful to understand the aloin molecular vibrational frequency. By comparing we choose the optimal experimental condition for water as solvent under alkaline conditions, the detection limit of the Aloin can reach a level of 5 ppm, which can be considered the theoretical basis for rapid detection of aloin content.