Carbon nanofiber/cellulose microfiber (CNF/CMF) hydrogel was prepared effectively with the help of ultra-sonication. The prepared CNF/CMF hydrogel was characterized by field emission scanning electron microscopy, Raman spectroscopy, X-ray photoelectron spectra, and elemental mapping. Then, CNF/CMF hydrogel modified screen printed carbon electrode (SPCE) was enforced for the electrochemical determination of nicotinamide adenine dinucleotide (NADH). Results of electrochemical studies reveal that the highly sensitive and discriminatory determination of NADH is effectively attained at CNF/CMF/SPCE. The designed sensor has a wide working range of 0.12-5100 mu M and with ultra-low detection limit was estimated to be 21 nM. Noteworthy, the electrode exhibits excellent selectivity from the potentially interfering biological compounds such as ascorbic acid, dopamine, and uric acid. These results suggest that this proposed electrochemical sensor can provide a promising detection for the simultaneous trace analysis of NADH in biological samples. (C) 2019 The Electrochemical Society.