Zinc (Zn) is an essential micronutrient that is generally deficient in soils to accommodate plant needs. Therefore, it is imperative to enhance the concentration and availability of Zn in soils and plants through fertilization. Currently, however, the binding characteristics and fate of Zn in soils under various fertilization regimes remain unclear. Here, two-dimensional correlation spectroscopy (2DCOS) and synchrotron-radiation-based spectro-microscopies were used to identify functional groups responsible for Zn binding in soils following different fertilization treatments. The results showed that a 23-year long-term manure application significantly increased the concentrations of Zn in both soil and corn grain as compared to those having received a traditional inorganic NPK treatment. The 2DCOS analysis of FTIR spectra showed that aliphatic C mainly contributed to Zn binding in the NPK-treated soils, whereas Si-O groups played a dominant role for Zn binding in the manure-treated soils. In soil dissolved organic matter, Zn²⁺ was first bound with Fe-O, followed by aliphatic C and polysaccharide C in the NPK treatment, but bound with aliphatic C-H prior to Si-O in the manure treatment. Furthermore, synchrotron-radiation-based FTIR and μ-XRF spectromicroscopies indicated that the distribution of Zn, clay minerals, sesquioxides and C functional groups was heterogeneous at the micro-scale, suggesting a heterogeneous binding site for Zn in soils. Using μ-XRF spectromicroscopies, a strong spatial correlation at the submicron scale between Fe and Zn was found in soils, suggesting that Fe-bearing minerals, likely Fe (hydr) oxides, contributed binding sites for Zn. Together, these results indicate that combining 2DCOS and synchrotron-radiation-based spectromicroscopies yields useful information for exploring the binding mechanisms among micronutrients, minerals and organic components in soils, particularly revealing how binding sites for micronutrients in soils can be modified according to fertilization regime.