Ions are the crucial signaling components for living organisms. In cells, their transportation across pore-forming membrane proteins is vital for regulating physiological functions, such as generating ionic current signals in response to target molecule recognition. This ion transport is affected by confined interactions and local environments within the protein pore. Therefore, the pore-forming protein can efficiently transduce the characteristics of each target molecule into ion-transport-mediated signals with high sensitivity. Inspired by nature, various protein pores have been developed into high-throughput and label-free nanopore sensors for single-molecule detection, enabling rapid and accurate readouts. In particular, aerolysin, a key virulence factor of , exhibits a high sensitivity in generating ionic current fingerprints for detecting subtle differences in the sequence, conformation, and structure of DNA, proteins, polypeptides, oligosaccharides, and other molecules. Aerolysin features a cap that is approximately 14 nm wide on the side and a central pore that is about 10 nm long with a minimum diameter of around 1 nm. Its long lumen, with 11 charged rings at two entrances and neutral amino acids in between, facilitates the dwelling of the single analyte within the pore. This characteristic enables rich interactions between the well-defined residues within the pore and the analyte. As a result, the ionic current signal offers a unique molecular fingerprint, extending beyond the traditional volume exclusion model in nanopore sensing. In 2006, aerolysin was first reported to discriminate conformational differences of single peptides, opening the door for a rapidly growing field of aerolysin nanopore electrochemistry. Over the years, various mutant aerolysin nanopores have emerged, associated with advanced instrumentation and data analysis algorithms, enabling the simultaneous identification of over 30 targets with the number still increasing. Aerolysin nanopore electrochemistry in particular allows time-resolved qualitative and quantitative analysis ranging from DNA sequencing, proteomics, enzyme kinetics, and single-molecule reactions to potential clinical diagnostics. Especially, the feasibility of aerolysin nanopore electrochemistry in dynamic quantitative analysis would revolutionize omics studies at the single-molecule level, paving the way for the promising field of single-molecule temporal omics. Despite the success of this approach so far, it remains challenging to understand how confined interactions correlate to the distinguishable ionic signatures. Recent attempts have added correction terms to the volume exclusion model to account for variations in ion mobility within the nanopore caused by the confined interactions between the aerolysin and the analyte. Therefore, in this Account, we revisit the origin of the current blockade induced by target molecules inside the aerolysin nanopore. We highlight the contributions of the confined noncovalent interactions to the sensing ability of the aerolysin nanopore through the corrected conductance model. This Account then describes the design of interaction networks within the aerolysin nanopore, including electrostatic, hydrophobic, hydrogen-bonding, cation-π, and ion-charged amino acid interactions, for ultrasensitive biomolecular identification and quantification. Finally, we provide an outlook on further understanding the noncovalent interaction network inside the aerolysin nanopore, improving the manipulating and fine-tuning of confined electrochemistry toward a broad range of practical applications.