<p>The formation and cleavage of chemical bonds are two fundamental processes in chemistry, and the nature of these bonds determines the physical and chemical properties of a molecule. Real-time observation of chemical bonding at the single-molecule level offers insights into transient intermediates that are normally inaccessible via ensemble measurements. Protein nanopores, with their unique geometries, can be tailored into nanoreactors. Molecular bond-making and -cleavage at the reactive site of a protein nanopore’s interior wall can be visualized by monitoring ionic current changes. Therefore, nanopore-based techniques can enhance the understanding of complex binding kinetics and reaction mechanisms. In this Review we summarize recent advances in using biological nanopores as both single-molecule nanoreactors and single-molecule biosensors. The discussion covers the kinetics of single-molecule reactions under nanopore confinement, the strategies for designing biological nanopores and the latest progress in revealing reaction intermediates and pathways at the single-molecule level. Finally, we emphasize unresolved challenges and anticipate future developments in this rapidly evolving field.</p><p></p>