The amino acid sequence of a protein encodes information on its three-dimensional structure and specific functionality. De novo protein design has emerged as a method to manipulate the primary structure for the development of artificial proteins and peptides with desired functionality. This paper describes the de novo design of a pore-forming peptide that has a β-hairpin structure and assembles to form a stable nanopore in a bilayer lipid membrane. This large synthetic nanopore is an entirely artificial device with practical applications. This peptide, named SV28, forms nanopore structures ranging from 1.6 to 6.2 nm in diameter assembled from 7 to 18 monomers. The nanopore formed with a diameter of 5 nm is able to detect long double-stranded DNA (dsDNA) with 1 kbp length. Moreover, the larger sized nanopore can discriminate and human telomeric DNA (G-quadruplex, G4). The blocking current signals allowed us to investigate the translocation behavior of dsDNA or G4 structure at the single molecule level. Such de novo design of peptide sequences has the potential to create novel nanopores, which would be applicable in molecular transporter between across lipid membrane.