"Electrochemical and Fluorescent Biosensors: Novel Strategies, Methods, and Materials" explores cutting-edge developments in biosensing technologies, focusing on electrochemical and fluorescent methods. This Special Issue delves into innovative strategies and methodologies that enhance the sensitivity, selectivity, and overall performance of biosensors. Contributors to this reprint present comprehensive research on novel materials, including nanomaterials and biomolecules, which play a crucial role in the advancement of biosensor technology. The reprint covers a wide range of applications, from medical diagnostics to environmental monitoring, showcasing the versatility and impact of these biosensors. Detailed discussions on the design, fabrication, and optimization of biosensors provide valuable insights for researchers and practitioners in the field. By integrating the latest advancements in electrochemistry and fluorescence, this reprint offers a holistic view of the current trends and future directions in biosensor development. This Special Issue serves as a valuable resource for those seeking to understand the complexities of biosensor technology and its practical applications. It highlights the collaborative efforts of scientists working towards more efficient and reliable biosensing solutions, emphasizing the importance of interdisciplinary approaches in achieving technological breakthroughs.