The Cu1−xNixFe2O4 nanoparticles (with x = 0, 0.3, 0.5, 0.7, and 1) were synthesized by using spray co-precipitation method at annealing temperature Ta = 900 °C in air for 5 h. The crystal structure, microstructure, oxidation state, and magnetic properties of the samples were characterized by using X-ray diffraction, synchrotron X-ray diffraction, scanning electron microscopy, X-ray absorption spectroscopy, and vibrating sample magnetometer. It was shown that all the samples have cubic structure. Lattice constant and grain size decrease, while the Curie temperature TC increases with increasing of Ni2+ content. A small amount of Fe2+ was found in all the samples. Cation distribution was determined by using a combination of magnetization measurements, extended X-ray absorption fine structure analysis, and Rietveld refinement from synchrotron X-ray diffraction data. It was indicated that Ni2+ ions occupy in octahedral site only, while Cu2+ ions distribute in both tetrahedral and octahedral sites. The variation of magnetic parameters is discussed based on Ni2+ concentration, grain size, the cation distribution, surface effect, and the presence of Fe2+ ion in the samples.