One Health is a comprehensive and cohesive strategy that seeks to effectively manage and enhance the well-being of people, animals, and ecosystems in a sustainable manner. Electrochemical biosensors are indispensable vectors for One Health management, with diverse applications across industrial process control, environmental monitoring, healthcare, microbiology, and quality assurance. The integration of nanotechnology has propelled innovative sensing algorithms, enhancing the efficiency and precision of biosensors. Nanoscale material integration amplifies their ability to detect molecules and single biomolecules with nano-biosensors utilizing nanoparticles, nanowires, carbon nanotubes (CNTs), nanorods, and quantum dots. Nanomaterials, distinguished by surface functionalities, tunability of physical/chemical properties, high detection sensitivity, large surface area, stability, and thermal/electrical conductivity, are instrumental in architecting nano-biosensors. The main limitations in current times are scalability, cross-sensitivity, complex immobilization pathways, and the toxicity factor of nanosensors, which are hazardous for the ecosystem as well as humans. This review explores the fundamentals of nanotechnology-enabled electrochemical biosensors, encompassing their composition, manufacturing materials, and diverse applications, highlighting their necessity in advancing biosensor technology to control environmental contamination/human diseases in order to manage One Health.