In situ piezoelectric FTIR (Fourier transform infrared) spectroelectrochemistry, a combination technique of in situ FTIR and electrochemical quartz crystal microbalance (EQCM), was used to study the electrochemical oxidation of 3,31,5,5'-tetramethylbenzidine (TMB) and the effect of some large-sized anions on the oxidation process. A V-shaped frequency response curve was observed during the electrochemical oxidation of TMB. The formation of TMB cation free radical and charge transfer complex (CTC) were experimentally found in the process of TMB electro-oxidation. In cyclic voltammetric test, the deposition of CTC onto the electrode surface from TMB solution was observed after several scanning cycles, even though the CTC is dissolvable in the solution. Further investigations suggested that the CTC was formed by TMB and TMB2+. Some representative large-sized anions, such as heparin, alizarin red and DNA, can affect the electro-oxidation of TMB. (C) 2008 Published by Elsevier B.V.