Different carbon-based materials have been compared for the development of NADH sensors: glassy carbon electrodes (GCE), multiwalled carbon nanotubes (MWCNT), and carbon black (CB). The GCE and MWCNT has been subjected to oxidative pretreatment to study the influence of oxidative groups for NADH oxidation. The materials had been characterized by FT-IR to identify the surface composition. The response of bare (GC) and GC/modified electrodes toward potassium ferricyanide have been employed to obtain information about the electroactive area and electron transfer rate. Studies of NAD + /NADH redox behavior showed that MWCNT and GCE exhibit high degree of passivation while CB shows no fouling effects. Catalytic effect of surface-oxygenated groups was also proved for GCE and MWCNT, and both, O-GCE and O-MWCNT, exhibited a lower oxidation overpotential compared to the respective untreated materials. Chronoamperometric quantification showed a linear dependence between 2–18 μ mol·L −1 and a detection limits of 6.2 μ mol·L −1 (GCE), 5.4 μ mol·L −1 (O-GCE), 3.2 μ mol·L −1 (GCE/CB), 9.6 μ mol·L −1 (GCE/MWCNT), and 4.9 μ mol·L −1 (GCE/O-MWCNT) were obtained. The analytical performances suggest that a careful choice of the material for NADH sensing is necessary depending on the sensor application.