Agricultural biomass wastes are an abundant feedstock for biorefineries. However, most of these wastes are not treated in the right way. Here, corn stalks (CSs) were assigned as the raw material to produce cellulose nanofibers (CNFs) via in situ Fenton oxidation treatment. In order to probe the formation mechanism of an in situ Fenton reactor, the bonding interaction of hydrated Fe 2+ ions and fiber has been systemically studied based on adsorption experiments, IR spectroscopy, density functional theory (DFT) calculations, and Raman spectroscopy. The results indicate that the coordination of the hydrated Fe 2+ ion to the fiber generates a quasi-octahedral-coordinated sphere around the Fe center. The Jahn−Teller distortion effect of the Fe center promotes the Fe−O 2 H 2 bonding interaction via reduction of the energy gap of the d z 2 orbital of the Fe center and π 2py /π 2pz orbitals of the H 2 O 2 molecule. The oxidation treatment of the pretreated CS by the in situ Fenton process shows the formation of a new carboxyl group on the fiber surface. The scanning electron microscopy image shows that the Fenton-treated fiber was scattered into the nanosized CNFs with a diameter of up to 50 nm. Both experimental and theoretical studies show that the pseudo-first-order kinetic reaction could describe the in situ Fenton kinetics well. Moreover, the proposed catalytic cycle shows that the large thermodynamic barrier is the cleavage of the O−O bond of H 2 O 2 to generate the • OH radical, and the whole catalytic cycle is found to be spontaneous at room temperature.