The aim of this study was mainly to develop a microbial biosensor for the simultaneous determination of lactic acid and pyruvic acid. In developing biosensor, lyophilised Lactobacillus delbruecki sp. bacterial cells were immobilised with polypyrrole on a platin electrode surface using electropolymerization method. Lactate concentration was determined based on the differences in amperometric responses at cathodic peak (+0.2 V) of potassium ferricyanide, whereas pyruvate concentration was determined using the differences at anodic peak (+0.1 V). The response of biosensor showed linearity between 0.1 and 1.0 mM for both of two substrates. Optimisation studies were carried out for amount of microorganism, pyrrole concentration, pH and temperature. In the characterisation studies, substrate specificity, interference effect of some substances on the biosensor response, and storage stability were established. (C) 2014 Elsevier Ltd. All rights reserved.