As a unique technique at the singe-molecule level to explore the distribution and temporal order of events, nanopore technology has attracted increasing attention. In comparison to the previous applications in DNA sequencing, this Focus Review highlights the technical details of biological nanopores, especially alpha-hemolysin, in the analysis of peptides and proteins. The instrument configurations, experimental interferences, and data analysis including the conformation of peptides and proteins and their interactions for single-molecule detection are discussed.