【Objective】 Based on a long-term (26 years) fertilization field experiment in Qiyang, Hunan, China, investigations were made of influeces of long-term fertilization on Fenton-like reaction and soil C storage in red soil, with the aid of the techniques of high-performance liquid chromatography (HPLC), high flux sequencing, Synchrotron radiation Infrared microscopic imaging, X-ray photoelectron spectroscopy and in-situ microcosm incubation in soil pores varying in size (1 000 μm, 20 μm and 0.45 μm). 【Method】HO ^? was trapped by terephthalic acid (TPA) (non-fluorescent), of which fluorescent product, i.e., 2-hydroxylterephthalic acid (HTPA), was determined with a high-performance liquid chromatographer (HPLC); Soil H₂O₂ quantified by a modified Fe²⁺-xylenol orange assay; Structure of bacterial communities and diversity analyzed with 16S ribosomal deoxyribonucleic acid (rDNA) gene sequencing, and interfacial processes identified with synchrotron radiation infrared microscopic imaging and X-ray photoelectron spectroscopy (XPS). In the microcosm incubation experiment, Polyvinylchlorid (PVC) cylinders were used and covered with a replaceable 0.45, 20, or 1 000 μm mesh fabric panel separately that allowed nothing but microorganisms and/ or roots to grow into the test cylinders. 【Results】Concentrations of H₂O₂, HO ^? and Fe(II) were higher in Treatment M than in Treatment NPK, indicating that root excreta and microbes involved in regulating the concentrations. Meanwhile, a linear relationship between H₂O₂ and HO ^? was found. H₂O₂ buffering capability was higher in Treatment M than in Treatment NPK, suggesting that the soil under long-term application of NPK degraded due to depletion of soil organic matter. Besides, the richness and diversity of soil microbes were significantly higher in Treatment M than in Treatment NPK. Furthermore, the effects of plant roots and microorganisms on soil bacterial community structure were lower in Treatment M than in Treatment NPK. In Treatment M soil bacterial communities affected the release of soil pH, DOC, soluble Fe and CO₂, while in Treatment NPK they induced Fenton reactions through production of H₂O₂ and reduced Fe(II). On root-soil interface, distribution of clay minerals (3 619 cm^-1), aliphatic C (2 914 cm^-1), carboxylic C (1 725 cm^-1) and hydroxyl C (1 135 cm^-1) varied sharply in pattern, and the presence of roots increased the proportion of Fe(II) on the interface. 【Conclusion】These findings suggest that long-term manure inputs to soil initializes free-radical reaction by activating microbial communities and mobilizing iron, which is conducive to soil C stabilization and storage by increasing recalcitrance and interactions of SOC. In addition, microbe-mediated Fenton-like reaction may affect C storage and soil fertility.