The electrochemical oxidation of dihydronicotinamide adenine dinucleotide (NADH) to the corresponding oxidised form (NAD⁺) has received considerable attention in the bio-electro-analytical field, owing to its significance as a substrate for the dehydrogenase based biosensors. This review highlights the principles and the recent developments of NADH amperometric detection using nanostructured material, chemically modified sensors and NADH oxidase based biosensors. The latest developments, including recent papers from the authors, are discussed with particular emphasis on the development of NADH/NAD⁺ dependent biosensors. Analysis of selected applications of NADH sensing and bio-sensing in biomedical, food and environmental areas are also given.