Here, we explore the possibility of using peptide-based materials as a membrane in solid-state nanopore devices in an effort to develop a sequence-specific, programmable biological membrane platform. We use a recently developed tyrosine-mediated self-assembly peptide sheet. At the air/water interface, the 5mer peptide YFCFY self-assembles into a uniform and robust two-dimensional (2D) structure, and the peptide sheet is easily transferred to a low-noise glass substrate. The thickness of the peptide membrane can be adjusted to approximately 5 nm (or even to 2 nm) by an etching process, and the diameters of the peptide nanopores can be precisely controlled using a focused electron beam with an attuned spot size. The ionic current noise of the peptide nanopore is comparable to those of typical silicon nitride nanopores or multilayer 2D materials. Using this membrane, we successfully observe translocation of 1000 bp double-stranded DNA with a sufficient signal-to-noise ratio of ∼30 and an elongated translocation speed of ∼1 bp μs-1. Our results suggest that the self-assembled peptide film can be used as a sensitive nanopore membrane and employed as a platform for applying biological functionalities to solid-state substrates.