In the last few decades, single-molecule techniques have emerged as transformative tools for exploring biological problems. By observing and analyzing individual molecules, these methods make it possible to investigate fundamental dynamics of biomolecular processes deeper. Unlike traditional ensemble methods that average the behavior of populations, single-molecule approaches provide a unique window to observe molecular heterogeneity, transient interactions, and dynamic processes that are otherwise hidden. This special issue brings together six mini-reviews that present how these cutting-edge methodologies are advancing our understanding of diverse and complex biological systems. Each review highlights unique applications, significant breakthroughs, and ongoing challenges. They collectively demonstrate the versatility and impact of single-molecule techniques. [BMB Reports 2025
58(1): 1-1].