A new type of polyethyleneimine‐protected copper nanoclusters (PEI‐CuNCs) is favorably developed by a one‐pot method under mild conditions. The obtained PEI‐CuNCs is characterized by X‐ray diffraction, X‐ray photoelectron spectroscopy, transmission electron microscopy, Fourier‐transform infrared (FTIR) spectroscopy and other techniques. It is worth noting that the proposed PEI‐CuNCs demonstrate a selective response to chromium(VI) over other competitive species. Fluorescence quenching of PEI‐CuNCs is determined to be chromium(VI) concentrations dependence with a low limit of detection of 8.9 nM. What is more, the as‐developed PEI‐CuNCs is further employed in building a detection platform for portable recognition of chromium(VI) in real samples with good accuracy. These findings may offer a distinctive strategy for the development of methods for analyzing and monitoring chromium(VI) and expand their application in real sample monitoring.