The WO(3) nanoparticles film is first employed as a support matrix for confining cytochrome c (cyt. c), an excellent model for studying electron transfer between the redox enzymes and the electrode. The surface pK(a) of nanostructured WO(3) film is estimated to be approximately 2.74 using electrochemical method. The present WO(3) surface with negative charge at the neutral solution is very benefit for the adsorption of cyt. c with positive charge and facilitates electron transfer of cyt. c. As a result, direct and fast electron transfer of cyt. c is realized at the nanostructured WO(3) surface with the redox formal potential (E(0)') of -133.5+/-1.7 mV (n=4) versus Ag/AgCl and heterogeneous electron transfer rate constant of 5.57+/-0.54 s(-1). Experimental data indicate that cyt. c is stably confined onto the WO(3) nanoparticles film, possibly due to the electrostatic interaction between WO(3) nanostructures and cyt. c, and processes its enzymatic activity toward H(2)O(2). Based on these results, the third-generation biosensor for H(2)O(2) is developed with high selectivity, free from not only common anodic interferences like ascorbic acid, uric acid, 3,4-dihydroxyphenylacetic acid, and so on, but also cathodic interference-O(2). The remarkable analytical advantages, as well as the characteristic of WO(3) nanoparticles film such as biocompatibility, low-cost, and facile to miniature give a strong basis for continuous, on-line detection of H(2)O(2) under pathophysiological conditions.