Electrochemical enzymatic biosensors represent a promising, low-cost technology for point-of-care (POC) diagnostics that allows fast response and simple sample processing procedures. In this review, we summarize up-to-date literature on NAD⁺/NADH (β-nicotinamide adenine dinucleotide)-dependent electrochemical dehydrogenase biosensors and highlight their applications in human physiological fluids. A brief comparison of various enzyme immobilization procedures is first presented, discussing preparation processes and principal analytical performance characteristics. In the following section, we briefly discuss classes of biosensors based on redox mediators-mediated electron transfer systems (METs). Finally, the conclusion section summarizes the ongoing challenges in the fabrication of NAD⁺-dependent electrochemical dehydrogenase biosensors and gives an outlook on future research studies.