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Although blood and cerebrospinal fluid remain the clinical gold standards, increasing attention is being directed toward non-invasive biofluids, such as urine, saliva, tears, and sweat. As accessible and patient-friendly alternatives. These biofluids contain diverse biomolecular signatures, including nucleic acids, proteins, metabolites, and microbial components, offering significant potential for continuous and real-time health monitoring. Recent advances in nanotechnology have transformed biomarker detection by enabling highly sensitive, selective, and rapid analytical platforms. This review provides an overview of key biomarkers associated with major diseases, including cancer, diabetes mellitus, and neurodegenerative disorders, with a focus on their presence in non-invasive biofluids. It further highlights recent progress in nanomaterial-enabled sensing strategies and the role of advanced nanostructures in enhancing detection performance. In addition, the convergence of wearable sensing technologies with artificial intelligence frameworks for continuous biomarker monitoring and intelligent disease management is also examined. 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