In the study, the preparation and electrochemical characterization of screen-printed electrodes (SPE) modified with 4’-mercapto-N-phenylquinone diamine (NPQD) and their behavior as electrocatalysts toward the oxidation of NADH with high stability are described. In particular, NPQD on the SPE substrate was deposited via electro-polymerization. Thus, the modified electrode exhibited a limit of detection (LOD) corresponding to 3.5 µM LOD with a sensitivity corresponding to 0.0076 ± 0.0006 µM/µA in the mouse serum. In all cases, NADH oxidation occurred at potentials approximately corresponding to 0.7 V vs Ag/AgCl electrode, and this indicates a decrease in the overpotential. We used the observations as a point of departure and developed an NADH biosensor based on the electrocatalytic oxidation of NADH. The applicability of the NADH sensor was demonstrated for the first time in 1) selectivity/sensitivity test via complex electrolyte as cell culture medium and 2) inflammatory and fibrotic responses by polyhexamethylene guanidine phosphate via NADH sensing in mouse serum.