Glycolytic metabolism of cells produces protons that are removed from the cytosol by transport proteins to create a pH difference between the adjacent bulk solution and the cell membrane surface. Therefore, tissue cells have distinct surface pHs because of varied glycocalyx and proton production capability. In this study, we proved the role of cell surface pH in peptide–cell interaction and peptide activation using lytic peptides with pH-dependent activity as probes. Properly, selected peptides could sense the specific pH zones on cells and thus demonstrated varied activity to tissue cells with different surface pHs. For a specific cell, the activity of pH-sensitive peptides changed accordingly as the cell surface pH was tuned up or down by proton channel regulators. Mechanistic studies revealed that cell surface pH directly affected peptide insertion into membranes by altering the secondary structure and aggregation status of membrane-bound pH-sensitive peptides. A pH-sensitive lytic peptide-designed based on the cell surface pH difference between a normal–cancer cell pair showed good selectivity to cancer cells. Therefore, cell surface pHs may present new opportunities to design therapeutic peptides with high cell specificity and selectivity.