Here we report on a facile, rapid, selective, and stable electrochemical sensing platform for nicotinamide adenine dinucleotide (NADH) based on polythionine-methylene blue/electrochemically reduced graphene oxide modified glassy carbon electrode (PTH-MBDES-ERG/GCE). The ERG was directly formed on a glassy carbon electrode (ERG/GCE) through one-step electrochemical reduction of GO in PBS solution (10 mM, pH 7.4). In a PBS electrolyte, respectively, containing 50% (v/v) three different deep eutectic solvents (DES), thionine and methylene blue were polymerized on the surface of ERG/GCE. The electrochemical properties of the polymers polymerized in different electrolytes were investigated. The PTH-MBDES-ERG composite has a higher performance in the electrocatalytic oxidation of NADH than that of PTH-MBDES. The as-prepared PTH-MBDES-ERG/GCE demonstrated excellent direct electrocatalytic oxidation toward NADH, providing a favorable platform for electron transfer from NADH to the electrode. The sensor displayed a wide linear range from 1.52 μM to 3333.33 μM with a limit of detection of 0.51 nM. The interference from the ascorbic acid and uric acid was negligible. The prepared sensor was further tested for the determination of NADH in artificial urine samples, showing the PTH-MBDES-ERG/GCE formed by electropolymerization have promising biomedical applications.