SP1 protein as a new type of biological nanopore is described and is utilized to distinguish single-stranded DNA at the single-molecule level. Using the SP1 nanopore to investigate single molecule detection broadens the existing research areas of pore-forming biomaterials from unsymmetrical biological nanopores to symmetrical biological nanopores. This novel nanopore could provide a good candidate for single-molecule detection and characterization of biomaterial applications.