The study is devoted to X-ray fluorescence spectroscopy (XRF) features of micro- and nanosized powder mixtures of copper and nickel. XRF is a high accuracy method that allows for both qualitative and quantitative analysis. However, the XRF measurement error due to the size of the studied particles is not usually taken into account, which limits the use of the method in some cases, such as analysis of Ni-Cu mixtures and coatings. In this paper, a method for obtaining copper and nickel nanoparticles was investigated, and the XRF of powder compositions was considered in detail. The initial micro- and nanoparticles of copper and nickel were studied in detail using SEM, TEM, XRD, and EDX. Based on experimental data, calibration curves for copper-nickel powder compositions of various sizes were developed. According to the results, it was experimentally established that the calibration curves constructed for nanoscale and microscale powders differ significantly. The presented approach can be expanded for other metals and particle sizes.