Ionic current recordings using biological nanopores provide a sensitive platform for monitoring reactions at the single-molecule level. Here we show that single enzymes can be trapped inside the confined space of a Cytolysin A (ClyA) nanopore for extensive periods of time by the electroosmotic flow, without the need of complex immobilization strategies or covalent chemistry. Remarkably, the binding of ligands and conformational changes to the internalized proteins are mirrored by specific changes to the nanopore conductance, indicating that ClyA nanopores are amenable for single-molecule enzymology studies. However, certain enzymes escape the nanopore too quickly to be properly sampled. Here we show that the residence time of proteins inside the ClyA lumen can be controlled by preparing interlocked protein-nanopore rotaxanes. Alternatively, the electrophoretic and electroosmotic driving forces can be fine-tuned by the precise modulation of the charges of the internalized proteins. The immobilization of single proteins inside the ClyA lumen opens new possibilities for real-time and label-free enzymology or proteomics studies, and for the fabrication of low-cost and portable sensing devices for the detection of analytes with high specificity.