Reduced nicotinamide adenine dinucleotide (NADH)-detecting electrochemical sensors are attractive in monitoring and diagnosing various physiological disorders of NADH abnormalities. The NADH detection methods using conventional electrodes are challenging due to slow electron transfer and fouling effect. Interestingly, paper-based flexible and disposable electrodes (PE) are superior for sensing biomolecules through simple detection procedures with excellent sensitivity and selectivity. Herein, to construct a conducting polypeptide-modified paper electrode, initially, polytyrosine (PTyr) is synthesized from l-tyrosine N-carboxy anhydride through ring-opening polymerization, and PTyr is drop-coated on the PE. The PTyr-modified paper electrode (PMPE) demonstrated excellent electrochemical properties and facilitated the electrooxidation of NADH at a lower potential of 576 mV. The PMPE displayed a linear detection between 25 and 145 μM of NADH concentration, with a lower detection limit of 0.340 μM. Under ideal circumstances, the sensor developed displayed an excellent NADH detection capability without interference with the most common electroactive species, ascorbic acid. The PMPE facilitates good electrocatalytic activity toward NADH, which can also be employed as a substrate material for biofuel cells.