4-nitrophenol (4-NP), a phenolic compound with highly toxic and carcinogenic properties, has become a global concern. As a fluorescence probe, a rapid and sensitive fluorescence assay for 4-NP was developed using cysteine-protected copper nanoclusters (Cys-CuNCs). The Cys-CuNCs were prepared using ascorbic acid-capped copper nanoparticles (CuNPs) as a precursor and cysteine as an etching agent. The prepared Cys-CuNCs showed excellent fluorescence properties and stability solubility with the maximum excitation and emission peak at 370 nm and 464 nm, respectively. In the presence of 4-NP, the fluorescence of CuNCs was quenched effectively. Single-factor experiments were investigated to optimize the pH of the buffer and the reaction time for nitrophenol detection. The optimal conditions were as follows: phosphate buffer (10 mmol·L^-1, pH 8.0) as a working buffer, mixing 4-NP and CuNCs for fluorescence scan. A sensitive analysis method for nitrophenol was constructed by utilizing the variation of fluorescence intensity F₀-F/F₀ as abscissa and 4-NP concentration as ordinate. The fluorescence quenching rate of CuNCs showed a linear relationship with the concentration of 4-NP in the range from 1 to 100 μmol·L^-1 with the detection limit of 0.31 μmol·L^-1. Four other 4-NP homologues and eight other organics were selected as the interferent; all of them had almost no effect on the fluorescence of CuNCs, indicating that the method had good anti-interference ability. In addition, the strategy was successfully applied to detect 4-NP in lake water with satisfactory recoveries from 97.0%～101%. The relative standard deviation was 2.71%～3.60%, indicating that the method is accurate and could be used for 4-NP detection in actual samples.