Abstract: The present invention provides a nanopore single-molecule protein sequencer, which mainly includes a nanopore array chip system, an ultra-low current detection system and a data processing and construction system; the chip mainly includes a chip of peptide charge screening, a chip of amino acid sequence reading based on a series of specific nanopores and the like, the design principle of which is as follows: a series of biological nanopores with amino acid targeted identification are designed according to properties such as hydrophilicity and hydrophobicity, polarity, and chargeability of amino acids, and characteristic ion flow signals of amino acids forming a protein to be detected in nanopore are obtained one by one; characteristic information of a protein sequence in each of the nanopores is acquired by an arrayed ultra-low current measurement system; a standard model peptide sequence information base is used for identifying, correcting, integrating and reading amino acid sequences.