A facile method, Graphene Oxide (GO) modified electrochemically pre-anodized screen-printed carbon electrodes (SPCEs), was developed for the determination of nicotinamide adenine dinucleotide (NADH) in neutral aqueous solution. Compared with the bare SPCEs and the reduced Graphene Oxide (rGO) modified SPCEs (rGO/SPCEs), the GO/SPCEs exhibit greatly enhanced electrocatalytic oxidation toward NADH. The scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS) provide the substantial evidence for the formation of edge-plane sites on the surface of SPCEs, rGO/SPCEs and GO/SPCEs. The GO/SPCEs show the excellent electrochemical character for the determination of NADH in the range from 0.8 to 500 μM with a detection limit of 0.10 μM. The formation of oxidation product NAD + was confirmed by in-situ UV-Vis spectroelectrochemistry. It shows a great promise for the design of the disposable amperometric biosensor for NADH.