A biosensor is an analytical device containing immobilized biological materials (enzymes, antibodies, nucleic acids, hormones, organelles, or whole cells) that can specifically interact with an analyte and produce physical, chemical, or electrical signals that can be measured, and then report these data and use them for medical applications. Biosensors have transformed diverse fields, including medical diagnostics to diagnose infectious diseases, environmental monitoring, and food safety, by enabling rapid, sensitive, and selective detection of target analytes. This study provides a comprehensive review of biosensor technology, covering the fundamental principles and practical applications. Recent advances in biological techniques and instrumentation involving fluorescence tags for nanomaterials have increased the sensitivity of biosensors. The current challenges and future prospects are also discussed in this study.