Melittin is a membrane-active peptide with strong anticancer activity against various cancers. Despite decades of research, the role of the singular Trp in the anticancer activity and selectivity of melittin remains poorly understood. Here, we propose a theranostic solution based on the substitution of Trp19 with a noncanonical fluorescent amino acid (Dap^AMCA). The introduction of Dap^AMCA residue in melittin stabilized the helical structure of the peptide, as evaluated by circular dichroism spectra and molecular dynamics simulations. In vitro hemolytic and anticancer activity assays revealed that introducing Dap^AMCA residue in melittin changed its mode of action with the cell membrane, resulting in reduced hemolytic toxicity and an improved the selectivity index (SI), with up to a five-fold increase compared to melittin. In vitro fluorescence imaging of Dap^AMCA-labeled melittin (MEL^FL) in cancer cells demonstrated high membrane-penetrating activity, with strong nuclear and nucleolar localization ability. These findings provide implications for novel anticancer therapies based on Trp-substituted designs and nuclear/nucleolar targeted therapy.