<p>Single-molecule studies are increasingly critical across diverse scientific disciplines. While nanofluidics research has significantly advanced in the past decade, translating nanofluidic device fabrication into practical single-molecule analytical applications remains a significant challenge. This review explores the burgeoning field of single-molecule analytical chemistry utilizing nanofluidic devices. We delve into the fundamental mechanisms and technologies enabling single-molecule sampling, isolation, manipulation, detection as well as single-molecule analysis within well-defined nanofluidic channels, highlighting the unique advantages of ultrasmall confined spaces and resulting unusual effects of nanofluidic devices. Despite remarkable progress, critical challenges and promising opportunities abound in this emerging field. This review also discusses future directions for this promising area of single-molecule analytical chemistry based on nanofluidic devices.</p> Graphical abstract <p></p>