A proper understanding of hydrogen–trap interactions in materials is of considerable significance, as it holds the potential to provide promising solutions to the long-standing issue of hydrogen embrittlement. In the present study, we employed a novel integrated approach combining electro-oxidation (EO) technique and thermal desorption spectroscopy (TDS) to characterize both reversible and irreversible deuterium in Fe samples. The samples were deuterium-charged at 500 kPa and temperatures ranged from 25 °C to 500 °C. Deuterium retention was measured by TDS for samples with and without EO treatment. Experimental findings demonstrate that the EO technique not only accelerates the expulsion of spontaneously releasable deuterium but also efficiently removes the majority of non-spontaneously releasable deuterium. It is evidenced that the proportion of reversible deuterium in the non-spontaneously releasable deuterium fraction reaches as high as 70%. Furthermore, an illustrative energy level diagram concerning the different barriers depending on the trap sites was devised to elucidate the trapping and diffusion behaviors of deuterium. Correspondingly, the microstructural trap sites associated with reversible or irreversible states were discussed in detail. This work enhances our understanding of hydrogen-Fe material interactions, thereby strengthening the fundamental theories underlying hydrogen embrittlement.