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This work describes the electrochemical performance of a novel composite based on nickel tetrasulfonated phthalocyanine (NiTsPc), deoxyribonucleic acid from calf thymus (CT-DNA) and reduced graphene oxide (rGO) for the electroanalysis of reduced β-nicotinamide adenine dinucleotide (NADH), through electrocatalytic oxidation. The modified electrode was denoted as CT-DNA/NiTsPc/rGO. Fourier transform infrared and ultraviolet-visible spectroscopies were performed to characterize the composite material. The electrochemical performance of the composite for NADH oxidation was investigated by cyclic voltammetry (CV), chronoamperometry and differential pulse voltammetry (DPV). The CT-DNA/NiTsPc/rGO modified glassy carbon electrode (GCE) showed an excellent electrocatalytic activity for NADH oxidation with apparent electrocatalytic rate constant (k obs ) of 7.35 × 10 5 L mol -1 s -1 and linear response range for NADH from 1 up to 1350 µmol L -1 for n = 12 (r = 0.999). The proposed sensor shows sensitivity, detection limit and quantification limit of 0.014 µA L mol , respectively. The prepared sensor was further tested for the determination of NADH in artificial human urine samples, showing promising biomedical applications.