A cytochrome c (Cyt c) electrochemical biosensor constructed by immobilization of the biomolecule on a hybrid material consisting of the conducting polymer poly-(3-methylthiophene) (P3MT) and multi-walled carbon nanotubes (MWCNT) is reported. Cyt c was immobilized onto l-cysteine (l-Cys) self-assembled monolayer-modified P3MT/MWCNT/GCE at neutral pH, where positively charged Cyt c strongly interacts with l-Cys negatively charged sites. The electron transfer rate constant, k(s), was 0.49 +/- 0.01 s(-1), and the surface coverage of Cyt c, Gamma, (1.6 +/- 0.8) x 10(-11) mol cm(-2). Under the optimized conditions, the mediatorless biosensor exhibits good electrocatalytic activity towards H(2)O(2) reduction. The amperometric measurements of H(2)O(2) at an applied potential of 0.0 V in 0.05 M Tris-HCl buffer of pH 8.0 exhibited good repeatability (RSD = 3.1%, n = 10) and an acceptable reproducibility for the biosensor preparation (RSD = 8.1%, n = 5). The lifetime of the biosensor was evaluated. A linear calibration graph for H(2)O(2) (r = 0.998) was obtained between 0.7 and 400 mu M, with a detection limit of 0.23 mu M. The analytical characteristics for H(2)O(2) determination were found to be better than those reported for other nano-structured cytochrome c biosensors. The apparent Michaelis-Menten constant for the reaction between Cyt c and H(2)O(2) was 451 mu M. The developed Cyt c/l-Cys/P3MT/MWCNT/GCE biosensor was applied for the biocatalytic determination of nitrite through the oxidation of NO(2)(-) to NO; by electrogenerated [Fe(4+)-Cyt c] species. Amperometry in stirred PBS solutions at a potential of +0.9 V allowed nitrite to be determined in a linear range between 10 and 100 mu M, with a detection limit of 0.5 mu M. (C) 2010 Elsevier B.V. All rights reserved.