For the first time a novel, simple and facile approach is described to construct highly stable glucose oxidase (GOx) multilayer onto glassy carbon (GC) electrode using thiourea (TU) as a covalent attachment cross-linker. The layer by layer (LBL) attachment process was confirmed by cyclic voltammetry, electrochemical impedance spectroscopy and Fourier transform infrared reflection spectroscopy (FT-IR-RS) techniques. Immobilized GOx shows excellent electrocatalytic activity toward glucose oxidation using ferrocenemethanol as artificial electron transfer mediator and biosensor response was directly correlated to the number of bilayers. The surface coverage of active GOx per bilayer, heterogeneous electron transfer rate constant (k(s)) and Michaelis-Menten constant (K-M), of immobilized GOx were 1.50 x 10(-12) mol cm(-2), 9.2 +/- 0.5 s(-1) and 3.42(+/-0.2)mM, respectively. The biosensor constructed with four-bilayers of TU/GOx showed good stability, high reproducibility, long life-time, fast amperometric response (5 s) with the high sensitivity of 5.73 mu A mM(-1) cm(-2) and low detection limit of 6 mu M at concentration range up to 5.5 mM. (C) 2011 Elsevier Ltd. All rights reserved.