Adsorption to soil minerals and occlusion within soil aggregates are two critical mechanisms to shield soil organic carbon (SOC) from microbial decomposition. Here, we combined the synchrotron radiation based Fourier transform infrared (SR-FTIR) spectroscopy, synchrotron radiation based micro X-ray fluorescence microscopy (μ-XRF) and two-dimensional correlation spectroscopy (2DCOS) analysis to in situ visualize the interiors of intact microaggregates from a typical Ferralic Cambisol in China, which had endured 25-year organic fertilization. Results showed that the spatial distribution and correlation between clay clusters and biopolymers were heterogeneous and significant, and also demonstrated that clay clusters were associated as nuclei with the potential of binding carbon at the submicron scale. Furthermore, the combination of SR-FTIR mapping and 2DCOS analysis could explore the strategy of identifying overlapped spectra and quantifying the sequestration reactivity for the first time. Specifically, carbon retention correlated as the binding sequence orders: 3630 cm −1 > 3610 cm -1 , 985 cm -1 > 898 cm -1 , indicating that Fe/Al oxyhydroxides and phyllosilicates could regulate the organic matter sequestration without the influence of spatial perturbations. Together, we conclude a combined methodology to assess the heterogeneous binding microenvironments between the mineral assemblages and biopolymers, which could also contribute to understand the process of carbon sequestration interrestrial ecosystems.