3-nitrotyrosine (3-NT) serves as a definitive biomarker of oxidative stress and assumes an essential role in the pathogenesis of diseases, so its detection is crucial for the diagnosis of diseases. Herein, we constructed a dual-emission ratiometric fluorescence probe with copper nanoclusters (CuNCs) and rhodamine B (RhB) as the response and reference signals. In the presence of 3-NT, the fluorescence intensity of CuNCs was significantly quenched through static quenching effect (SQE) and inner filter effect (IFE), whereas the fluorescence intensity of RhB kept unchanged. Notably, the sensor achieves high stability, sensitivity, and selectivity for 3-NT detection. Moreover, a portable sensing platform integrated with a 3D printer and a smartphone was constructed to fulfill the need for convenient measurement. The sensor was subsequently employed to determine 3-NT in both human serum and urine specimens, yielding results that were deemed satisfactory. The probe holds broad application potential and provides some ideas for the fluorescence detection of other biomarkers.