An amperometric bacterial sensor with current response to Fe²⁺ and S₂O₃^2– ions has been designed by immobilizing an acidophilic biomass of Acidithiobacillus ferrooxidans on a multi disk flat-front oxygen probe. The bacterial layer was located between the oxygen probe and a membrane of cellulose. A filtration technique was used to yield the bacterial membranes having reproducible activity. The decrease of O₂ flow across the bacterial layer is proportional to the concentration of the dosed species. The dynamic range appeared to be linear for the Fe²⁺ ions up to 2.5 mmol L^–1 with a detection limit of 9 × 10^–7 mol L^–1 and a sensitivity of 0.25 A L mol^–1. The response of the biosensor is 84 s for a determination of 2 × 10^–4 mol L^–1 Fe²⁺. Optimizing the Fe²⁺ determination by A. ferrooxidans sensor was carried out owing to Design of Experiments (DOE) methodology and empirical modelling. The optimal response was thus obtained for a pH of 3.4, at 35 °C under 290 rpm solution stirring. S₂O₃^2– concentration was determined at pH 4.7, so avoiding its decomposition. The concentration range was linear up to 0.6 mmol L^–1. Sensitivity was 0.20 A L mol^–1 with a response time of 207 s for a 2 × 10^–4 mol L^–1 S₂O₃^2– concentration.