The development of methods to detect metal ions in water for maintaining water quality and diagnosing metal ion-related diseases is important for improving our quality of life. In this study, we report that we succeeded in selectively detecting metal ions in water, particularly Cu2+ ions, using fluorescent peptides. First, we prepared seven peptides C1–C7 with different sequences, each containing two pyrenylalanine units (amino acid with fluorescent dye pyrene in the side chain; Pyr) and 0, 1, or 2 His units in a 6-mer cyclic peptide by an Fmoc-based solid-phase peptide synthesis. These cyclic peptides also included a tail consisting of four Arg units to improve water solubility. The cyclic peptide C1 (cyc-Pyr-Gly-Gly-Pyr-Gly-Glu(CH2-CH2-CO-Arg4)-), which does not contain a His unit, did not show any fluorescence change when aqueous solution containing 17 metal ions were added. However, C2–C7 containing one or two His units showed a fluorescence change accompanied by quenching only when an aqueous solution containing Cu2+ was added. On the other hand, the linear version of the C1–C7 peptide showed a fluorescence response with quenching to multiple metal ions, indicating no selectivity for specific metal ions. The degree of quenching of C2–C7 caused by Cu2+ depended on the sequence of the cyclic peptide and the number of His units. For example, C2 (cyc-Pyr-His-Gly-Pyr-Gly-Glu(CH2-CH2-CO-Arg4)-) showed poor fluorescence response to Cu2+, while C7 (cyc-Pyr-Gly-His-Pyr-His-Glu(CH2-CH2-CO-Arg4)-) showed excellent fluorescence response to Cu2+. Furthermore, it was revealed that C7 was bound to Cu2+ in a 1:1 ratio through fluorescence titration curves and ESI-Mass spectra. These results indicate that the conformational restriction, the sequence of the peptide and the presence of His units influence the selective fluorescence detection of Cu2+, and demonstrate that peptides with appropriate structures can achieve the specific fluorescence detection of Cu2+.