Herein we report a NiO/nanoparticles (NiO/NPs) modified carbon ionic liquid paste electrode (IL/NiO/NPs/CPE) was fabricated and used to investigate the electrochemical behavior of NADH in aqueous solution. The proposed sensor was prepared by mixing hydrophilic ionic liquid 1-methyl-3-butylimidazolium bromide, NiO/NPs, graphite powder and liquid paraffin together. The oxidation peak potential of NADH at the surface of the ionic liquid NiO/NPs carbon paste electrode appeared at 635 mV, which was about 70 mV lower than the oxidation peak potential at the surface of traditional carbon CPE under similar conditions. On other hand, the oxidation peak current was increased to about four times at the surface of IL/NiO/NPs/CPE compared to CPE. The linear response range and detection limit were found to be 0.03–900 μmol L−1 and 0.009 μmol L−1, respectively. IL/NiO/NPs/CPE was successfully applied for the determination of NADH in real samples such as water, serum and urine. The results showed that the proposed method is highly selective, sensitive with a fast response for NADH analysis.