Abstract Objective The purpose of this study was to in situ characterize the demineralization (namely dissolution of hydroxyapatite) on the surface of the human enamel using electrochemical impedance spectroscopy (EIS) technology. Methods Fresh human third molars extracted without visible evidence of caries, were used in this study. After they were immersed in a demineralizing solution prepared from lactic acid and carboxy methyl cellulose sodium (Na-CMC) buffering at pH 4, demineralization happened on their surfaces. EIS of the specimens were measured at a series of immersed interval. X-ray diffractometer (XRD) were used to distinguish the microstructure of the surface layer of the specimens. The depositions that appeared in the demineralizing solution after 46 h immersion were analyzed by fourier transform infrared spectrometer (FTIR). Results XRD analysis revealed that the percentage of intensity ( I%) of HAP gradually decreased with the elapsed immersing time, which indicated the dissolution of HAP columns of enamel. Nyquist spectra were fitted with an equivalent circuit characterized by some parameters, such as Q and R p (error < 0.1%). Changes of the parameters’ values revealed that the rate of demineralization accelerated at the forepart of the demineralization, but slowed down beyond 70 h immersion. Conclusions The results suggested that EIS was proved to be a useful method for in situ investigating and in vivo detecting the demineralization of the enamel.