Peptides play important roles in a variety of biological processes. Here, we studied the transport of peptides containing mainly aromatic amino acids in protein pores engineered with aromatic binding sites. With an increase in the length of the peptide, both the event mean dwell time and the current blockage amplitude increased. The dissociation rate constants k(off) decreased significantly, while the association rate constants k(on) decreased slowly as the peptide length increased. Thus, the overall reaction formation constants K-f, and hence the binding affinities of various peptides to the protein pore, are largely dependent upon the dissociation rate constants rather than the association rate constants. Furthermore, in a protein channel modified with aromatic binding sites, aromatic amino acid components contributed more to the dwell time and current blockage of the events than other types of amino acids, although the van der Waals volumes of amino acids also affected the event signatures. The effect of protein structure on peptide translocation was also investigated. With more aromatic binding sites engineered inside the lumen of the protein pore, a stronger binding affinity between peptides and the pore was observed. With the much enhanced resolution of the engineered protein pore, a series of short peptides, including those differing by a single amino acid, was successfully differentiated and simultaneously quantified. In addition to providing a rapid and cost-effective method for the peptide detection, the engineered protein pore approach offers the potential for peptide and protein sequencing.