Biosensors for d-lactate and acetaldehyde were developed, based on screen-printed electrodes and NAD +-dependent dehydrogenases. Modification of screen-printed electrodes with the mediator Meldola Blue or with Meldola Blue-Reinecke salt resulted in sensitive, low cost and reliable NADH detectors. The biosensors were realised in two configurations, as disposable and reusable devices. Single-use sensors were obtained by simple deposition of enzyme and cofactor on the surface of mediator-modified electrodes. Chronoamperometry was used for the detection of substrates in small volumes of samples (25 μl). Immobilisation of dehydrogenases by entrapment in poly(vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ) allowed sensors to be obtained with sufficient operational stability. Amperometry in stirred solutions was the detection technique with biosensors for multiple use. The 3 σ detection limits for acetaldehyde were 1 μM by amperometry and 6 μM by chronoamperometry and for d-lactate-0.03 μM and 0.05 μM for reusable and disposable biosensors respectively. The biosensors were applied in the analysis of some French and Romanian wines.