Biosensing plays a vital role in healthcare monitoring, disease detection, and treatment planning. In recent years, nanofluidic technology has been increasingly explored to be developed into lab‐on‐a‐chip biosensing systems. Given now the possibility of fabricating geometrically defined nanometric channels that are commensurate with the size of many biomolecules, nanofluidic‐based devices are likely to become a key technology for the analysis of various clinical biomarkers, including DNA (deoxyribonucleic acid) and proteins in liquid biopsies. This review summarizes the fundamentals and technological advances of nanofluidics from the purview of single‐molecule analysis, detection of low‐abundance molecules, and single‐cell analysis at the subcellular level. The extreme confinement and dominant surface charge effects in nanochannels provide unique advantages to nanofluidic devices for the manipulation and transport of target biomarkers. When coupled to a microfluidic network to facilitate sample introduction, integrated micro‐nanofluidic biosensing devices are proving to be more sensitive and specific in molecular analysis compared to conventional assays in many cases. Based on recent progress in nanofluidics and current clinical trends, the review concludes with a discussion of near‐term challenges and future directions for the development of nanofluidic‐based biosensing systems toward enabling a new wave of lab‐on‐a‐chip technology for personalized and preventive medicine.