Microbial biosensors can be used to overcome the limitations of conventional BOD analysis. In the present study a biosensor was designed based on amperometric oxygen sensor, an easily replaceable biomembrane of cellulose acetate and immobilised cells of different microorganisms. Immobilised cells of Bacillus subtilis and Pseudomonas sp. were used in the study. Biosensor output signal depends on the concentration of substrate that indicates the organic pollution of waters. Biosensor output signal was analysed classically according to the steady-state method. The calibration of biosensor was based on the change of biosensor response between an initial value and the stable end-point of the signal. Studied biosensor can be used for the measurements of biochemical oxygen demandmore effectively in the concentration range of 30-80mg/L for bacillus subtilis sp. and 5-45 mg/L for pseudomonas sp.