A novel electrochemical biosensor integrating the strictly autotrophic bacterial strain Leptospirillum ferrooxidans as a recognition element and a Clark type oxygen probe as a transducer was designed, metrologically and analytically characterized and applied for the specific Fe(2+) determination. The bacterial Fe(2+) oxidation involves O(2) consumption, thus the quantification was performed registering the decrease of the oxygen reduction current. The limit of detection was found to be 2.4 micromol L(-1) and the sensitivity of the determinations-3.94 nAL micromol(-1). The response time of the biosensor is 18s for Fe(2+) concentrations of 10(-5) to 10(-4) mol L(-1). The biosensor was applied as well for the indirect determination of Fe(2+) oxidizing species such as Cr(2)O(7)(2-), reaching a sensitivity of 2.47 nAL micromol(-1). The transducer characteristics were evaluated and optimized to obtain short response time and high sensitivity. The analytical performances of the biosensor subject of the present work were found to be similar to that of the At. ferrooxidans based one developed by the authors earlier, avoiding however the sulfur compounds interference, because of the substrate specificity of the applied bacterial strain.