Calibration is one of the most important factors affecting the measurement accuracy of X-ray energy/wavelength-dispersive spectrometers (EDS/WDSs). In this work, three Cu content reference materials used in AlCu films for X-ray EDS/WDS calibration are prepared using magnetron sputtering deposition. The Cu content standard values of the three reference materials are determined using an inductively coupled plasma optical emission spectrometer (ICP-OES) and the uncertainties of the reference materials are evaluated. The results show that the homogeneity and stability of the three Cu content reference materials used in AlCu films meet the requirements. The Cu content standard values of the three reference materials are 2.56%, 14.64%, and 49.46% (mass fraction), respectively. Their extended uncertainties are 0.14%, 0.42%, and 0.98% (k = 2), respectively. Using quantitative analysis, it was determined that the three developed reference materials meet the calibration requirements of X-ray EDS/WDS, thus guaranteeing the accuracy and consistency of the measurement results.