Nanophase iron (np-Fe0) is a major product of space weathering and its presence significantly alters the reflectance spectral characteristics of lunar soil. Previous studies have established that the np-Fe0 particles originate from the reduction of ferrous ions in the plasma, in-situ decomposition of olivine and pyroxene, and disproportionation of ferrous ions in solid ferrosilicates. In this study, sample charging effects were eliminated and in situ nanoscale valence state analysis of iron-bearing phases in Chang’E-5 lunar soil was conducted by combining focused ion beam (FIB) microscopy, Auger electron spectroscopy (AES), and transmission electron microscopy-electron energy loss spectroscopy (TEM-EELS) techniques. The results indicate that the contents and valence states of iron in the np-Fe0 particles, amorphous matrix, and ferrosilicates differ. The np-Fe0 particles were found to be composed of pure metallic iron, whereas ferrous and ferric iron ions were present in olivine crystals and the amorphous matrix, respectively. The discovery of both metallic and ferric iron in the amorphous matrix of Chang’E-5 lunar soil offers new insights regarding the disproportionation reaction of Fe2+ on the lunar surface. This study demonstrates that the combination of FIB, AES, and TEM-EELS is an effective and precise approach for analyzing the valence states of iron-bearing phases in lunar soil, which can be extended to other extraterrestrial samples and other multivalent elements.