In this article, we constructed an electrochemical sensor for highly sensitive determination of β-nicotinamide adenine dinucleotide (NADH) using the poly(diallyldimethylammonium chloride)-functionalized reduced graphene oxide nanocomposite modified electrode (PDDA-rGO/GCE). The nanocomposite was characterized by transmission electron microscopy, Fourier transform-infrared spectrometry, ultraviolet-visible absorption spectrometry, cyclic voltammetry, and electrochemical impedance spectroscopy. The experimental results demonstrated that the fabricated sensor exhibited excellent electrocatalytic activity on the oxidation of NADH with a decreased about 200 mV in overpotential and 5.0-fold increment in the oxidation peak current compared to a bare GCE. Applying cyclic voltammetry, a wide linear range from 1.0 × 10 −7 to 2.9 × 10 −3 M with low limit of detection (LOD) of 3.4 × 10 −8 M and high sensitivity of 463.3 μA mM −1 cm −2 were obtained, which was superior to other NADH electrochemical sensors reported previously. Furthermore, the fabricated NADH sensor possessed excellent selectivity, reproducibility, and stability. The oxidation mechanism of NADH at the PDDA-rGO/GCE was also discussed in detail.