This study aimed to develop a label-free, sensitive, selective, and environment-friendly fluorescent peptide probe His-His-Trp-His (HHWH) for determining the concentration of copper ion (Cu2+) in aqueous solutions. The results demonstrated that the designed HHWH has a high selectivity and sensitivity for monitoring the concentration of free Cu2+ via quenching of the probe fluorescence upon a binding of Cu2+. The fluorescence intensity of the HHWH had a linear relationship with the concentration of Cu2+ between 10 nM and 10 μM, and the detection limit was 8 nM. Furthermore, HHWH could be regenerated with sulfide ions at least five times. The concentrations of Cu2+ in three different real water samples were detected using this probe, and the results were consistent with the one detected using an atomic absorption spectrometer. Thus, HHWH can be used as an accurate and feasible fluorescent peptide probe for detecting Cu2+ in aqueous solutions.