The effects of luteolin (LU) as a new mediator on the electrooxidation of the reduced nicotinamide adenine dinucleotide (NADH) were investigated and developed a new disposable NADH sensor. Firstly, screen printed carbon electrodes (SPCE) modified with gold nanoparticles (AuNP), and then, LU was deposited on SPCE modified with AuNP (SPCE/AuNP) using cyclic voltammetry. Electrochemical behavior of LU on SPCE/AuNP was investigated and, the redox proses of LU on SPCE/AuNP was found to be adsorption controlled. The number of cycles was optimized for the electrochemical deposition of LU and determined to be 15 cycles. LU modified SPCE/AuNP (SPCE/AuNP/LU) was found to show the electrocatalytic effect on electrooxidation of NADH and acts as a mediator. Working potential and pH were optimized for electrochemical detection of NADH with the proposed SPCE/AuNP/LU sensor and found to be +0.225 V and 7.25, respectively. The proposed NADH sensor showed a well linear response from 15.90 to 925 μM with a detection limit of 4.57 μM and a sensitivity of 11.19 μAmM −1 . The repeatability of the NADH sensor was tested +0.225 V with 50 µM NADH solution. The relative standard deviation (RSD) was calculated as 3.68% (for n=10). The operational stability studies have shown that the initial amperometric response of sensor to NADH decreased by 62.1% at the 30th day. The analysis of NADH in artificial human serum samples was successfully performed with SPCE/AuNP/LU sensor.