The redox state is not only a key factor in assessing planetary habitability, but also a crucial variable connecting Earth’s interior spheres and surface environment via mass recycling and co-variation. Iron (Fe), as the most abundant multivalent element in terrestrial planets, its oxidation state (expressed as Fe³⁺/ΣFe or Fe³⁺/Fe²⁺) being the most important parameter reflecting the oxygen fugacity of the system. This study thoroughly investigates the application and theoretical foundation of electron probe microanalysis (EP-MA) in the determination of Fe oxidation states in geological samples. We comprehensively analyzed the principles of X-ray emission and absorption, self-absorption effect, and crystal field theory, providing a detailed elucidation of the spectral characteristics of Fe’s L-series lines. Accordingly, we compared the advantages and disadvantages of the peak shift method and the flank method, and examined the damage inflicted on samples under electron bombardment. The results demonstrate that EPMA holds extensive application potential and overwhelming advantages in the determination of Fe oxidation state, but the analytical protocol and condition must be optimized to enhance test accuracy and reliability according to the characteristics of different samples. This research offers theoretical support and practical guidance for further development and promotion of Fe valence determination methods, holding significant implications for earth science research.