In this work, a novel amperometric biosensor of hydrogen peroxide (H2O2) was developed based on the immobilization of myoglobin (Mb) on the surface of the multi-walled carbon nanotube (MWCNT) -Nafion-cysteamine (CA) modified gold electrode (Au) and its electrocatalytic activity was used for the determination of nitrite (NO2-). In the optimization studies, the best MWCNT and myoglobin amount were investigated. It was discovered at the experiments for the optimization of the working conditions that the buffer at this study as 50.0 mM, pH 7.0 phosphate buffer (PBS) and working temperature as 30 degrees C for the H2O2 biosensor. It was determined at the characterization studies on the biosensor that linear results are obtained between the ranges of 0.1 mu M to 70.0 mu M for H2O2 concentration and 1-250 mu M for NO2-. The reproducibility of the biosensor was determined both H2O2 and nitrite. From the experiments, average value, standard deviation (SD) and coefficients of variation (CV%) were calculated to be 10.02 +/- 0.43 mu M, and 4.29% for 10.0 mu M H2O2 (n = 6) and 52.0 +/- 2.1 mu M, and 3.89% for 50.0 mu M nitrite (n = 8), respectively. At the same time the sample was analyzed for NO2- in drinking and mineral waters. (C) 2014 Elsevier B.V. All rights reserved.