In this study, a label-free sensing method based on a functionalized nanopipette is proposed for detection of alkaline phosphatase (ALP) activity. This method is based on the unique charge transport properties of nanopores in which the ionic current can be modulated by variation in the charge on the inner surface of the nanopipette. A phosphorylated peptide, O-phospho-l-tyrosine (p-Tyr), was used as a model substrate and to functionalize the nanopipette. The covalent functionalization procedures were characterized by measuring the current-voltage (Isingle bondV) curves and extracting the ionic current rectification status. On ALP catalysis, the removal of the p-Tyr phosphate group induces a decrease in the negative surface charge, subsequently leading to a sensitive response in the ionic current change. An ANOVA test confirmed a linear relationship between the normalized ionic current change and ALP activity. The functionalized nanopipettes demonstrated a directly measured detection limit of 0.1 mU/mL, and excellent selectivity against four common interfering proteins. Furthermore, this nanopore-based method has potential for use in characterizing or sensing the activities of other enzymes with a similar biochemical process to ALP.