The surface oxide species produced by electrochemical pretreatment of a glassy carbon electrode were characterized using in situ Fourier transform IR spectroscopy and electrochemical techniques in aqueous (H2O) and deuterated water (D2O) solutions. This study presents the first in situ IR evidence of the surface oxide species on glassy carbon electrodes in aqueous solution. The results showed that the potential-dependent frequency shift is about 30 cm-1 V-1 for quinone-like species in deuterated water. There is a distinctive potential dependence for carboxylic-like species. We also investigated the effect of potential changes on the surface composition (i.e. transformation between quinone and hydroquinone etc.). On the basis of the reflectance theory of the surface film on a moderate-adsorption substrate, the origin of the changes in the spectral band and potential-dependent relationship are discussed. The open-circuit potential decay results implied that the acid-base behaviour is similar to that of graphite oxide.