Electronic absorption spectra, fluorescence emission spectra, 1HNMR and ab initio quantum calculation are used to study the acid-base equilibrium of puerarin in cetytrimethylammonium bromide (CTAB) micelles with different microstructures and microenvironments. Experiments suggest the microenvironment provided by CTAB micelle cause the acid-base equilibrium of puerarin to move to the deprotonation reaction. The changes in the chemical shifts of the individual groups of protons in CTAB indicate that the location of puerarin changes from the inner to the outer of the CTAB micelles with an increase in puerarin concentration, which enhances the interaction between puerarin and CTAB. The binding of puerarin with CTAB micelle is a spontaneous (deltaG<0) and endothermic process (deltaH<0), and the hydrophobic and electrostatic force is the main driving force for its solubilization.