Zinc oxide Nanowires (ZnO-NWs) are promising biosensor materials and hold the key to overcoming challenges in the field. This chapter provides an introductory overview of biosensing technology, focusing on the fundamental principles and comparing ZnO-NWs with other nanostructures regarding the surface area, reactivity, electrical properties, charge transport behavior, optical, magnetic, and piezoelectric properties, and mechanical flexibility. Providing the synthesis and characterization methods, ZnO-NWs’ biosensing processes are also elaborated on surface modification for selectivity, integration with microfluidic systems, enhancing signal transduction, and connecting with biological elements like enzymes, antibodies, and DNA. The chapter also discusses the applications of ZnO-NWs-based biosensors in clinical diagnostics, environment monitoring, and agricultural and food safety. It investigates some case studies and challenges in practical deployments. It emphasizes how ZnO-NWs can address these challenges, such as stability, reproducibility, scalability, and integration with electronic devices. Adequate emerging trends that include the utilization of machine learning (ML) and artificial intelligence (AI) for the further enhancement of sensing performance and the prospects of ZnO-NWs in multifunctional sensor platforms are also presented, with an overview of how ZnO-NWs have been the focus for significant impacts on biosensing. Also presented in this chapter are potential breakthroughs and future research directions.