A new L-lactate selective impedimetric biosensor based on a lactate dehydrogenase (LDH)/pyruvate oxidase (PyrOx) bioselective membrane is reported, fabricated using enzyme drop coating and cross-linking via glutaraldehyde vapours. The sensing strategy is based on the measurement of the change in impedance caused by the generation of charged ions (CH3COO , H+ and HCO3 ), from PyrOx catalysed pyruvate oxidation, the pyruvate resulting from L-lactate oxidation catalysed by LDH + nicotinamide adenine dinucleotide (NAD(+)). The experimental conditions, pH and buffer concentration, were optimized for best biosensor response, and operational/storage stability and selectivity/specificity were investigated. The limit of detection for L-lactate, was 17 and 20 mu M, considering changes in resistance and charge separation parameters at and close to the LDH-NAD(+)/PyrOx interface, respectively. The biosensor was successfully employed for the determination of L-lactate in yogurt samples. (C) 2017 Elsevier Ltd. All rights reserved.