Nanofillers have emerged as versatile materials with immense potential in various biosensing and biotechnological applications, including tissue engineering, drug delivery, gene silencing, and biomedical imaging. This review explores the diverse types of nanofillers utilized in biosensors and biotechnological systems, their synthesis methods, classification, and their impact on enhancing the performance and functionality of biotechnological applications. The review delves into the intricate role of nanofillers in biosensors, investigating their influence on sensitivity, selectivity, and overall performance. It highlights their contributions to advancing diagnostic capabilities, biomarker detection, and real-time monitoring. Additionally, the review explores the integration of nanofillers in novel biosensing platforms, shedding light on their potential to revolutionize point-of-care diagnostics and personalized medicine. Further, discusses the challenges associated with nanofillers, such as toxicity and biocompatibility concerns, and provides insights into future directions and emerging trends in this rapidly evolving field. By comprehensively examining the synthesis, characterization, and performance enhancement strategies of nanofillers in multifarious biological applications. This review article aims to inspire further research and innovation for the development of advanced biotechnological systems.