In this work, a single-step reduction method for the synthesis of highly stable water-soluble copper nanoclusters (CuNCs) at ambient conditions in which Thunnus albacares fish protein (FP) played the dual role of a capping and reducing agent is reported. The physicochemical properties of the prepared CuNCs were characterized by using UV-Visible Spectroscopy, FTIR, TEM, and time-resolved fluorescence spectroscopic studies. The FP-CuNCs showed high photostability under different ionic strengths, pH, with good quantum yield. It showed maximum emission at 446 nm upon its excitation at 330 nm. The excellent fluorescent properties of FP-CuNCs were further utilized to detect Fe+3 ions in aqueous condition within the concentration range of 0–50 μM and the sensing assay exhibited a limit of detection of 0.68 μM. The mechanism involved in the fluorescence detection is ascribed to the synergistic static and IFE-induced fluorescence quenching of FP-CuNCs. The practical applicability of the detection probe was further established by carrying out Fe+3 ion detection in environmental sample analysis.