The presented study is focused on the development of repetitive, fast and versatile "on-off" signaling electrochemical genosensor for detection of pagA gene of Bacillus anthracis. Such a sensor will be further used in the portable devices dedicated to molecular diagnostic. The DNA molecular beacon probe was immobilized onto gold electrodes in its folded state through the alkanethiol linker at the 5'-end, while the 3'-end was labeled with methylene blue redox molecule. Initial optimization was conducted for HPLC purified cDNA. Nonetheless application of such prepared biosensor for unpurified PCR product required further investigations. As the sensor response was significantly dependent on the stem-loop architecture, the three different hairpin probes were analyzed. The detected sequence was interior to amplicon, identical as in case of cDNA. The developed "on-off" genosensor allows for detection, in 5 minute, the pagA gene sequence at the level of 5.7 nM with 22.9-86.0 nM linear detection range also in the great excess of other DNA presented in the sample. Both LOD and linear range were significantly improved in case of longer assay time. The biosensor proved its applicability also in detection of pagA gene of Bacillus anthracis in PCR samples amplified with reduced number of cycles. (C) 2018 The Electrochemical Society.