Pretreatment of steam-exploded poplar by Fenton oxidation was used to effectively biodegrade lignin. The structure of lignin samples (LS from steam-exploded poplar and LSF from samples further treated by Fenton oxidation) obtained by enzymatic hydrolysis during pretreatment were characterized and compared. The results showed that the demethoxy reaction occurred in the process of Fenton oxidation. GPC results indicated that the weight average molecular weight (Mw) of LSF did not change significantly, indicating that that there was no significant condensation during Fenton oxidation pretreatment. Heteronuclear single quantum coherence nuclear magnetic resonance (HSQC NMR) results suggested that the proportion of the three-unit basic structural units of poplar (H, G, and S) were significantly changed during Fenton oxidation process, and more lignin S units were removed than lignin G units. The content of β-O-4 linkages was lower in LSF (74.0%, as a fraction of β-O-4 + β-5 + β-β) than in LS (78.2%), which indicated that β-O-4 linkages were destroyed to a certain extent during Fenton oxidation process, and the content of total lignin interunit linkages including β-O-4, β−β, and β-5 linkages over total lignin aromatic subunits (Ar%) in LSF was higher, reaching 49.9%.