Formation of mineral-associated organic matters is essential for long-term soil organic carbon preservation. However, the regulation pathways of mineral-associated organic matters in soils remains largely undescribed, especially at the submicron scale. Using a well-controlled long-term (32 years) field experiment at the Jiangxi Institute of Red Soils, Jinxian, China, we showed that long-term manure fertilization significantly (P < 0.05) increased the available Al and Fe concentrations over one order of magnitude in soil water dispersible colloids compared to no fertilization and chemical fertilization. Nano-scale secondary ion mass spectrometry (NanoSIMS) images and region of interest (ROI) analysis provided direct evidence demonstrating that long-term manure fertilization increased the 27Al16O−/12C−, 27Al16O−/12C14N−, 56Fe16O−/12C−, and 56Fe16O−/12C14N− ratios compared to no fertilization and chemical fertilization. This finding revealed that long-term manure fertilization may potentially enhance the stability of organic carbon and nitrogen. Synchrotron radiation based FTIR spectromicroscopy further supported that secondary minerals (<950 cm−1) were co-localized with amines, lipids, and proteins in the long-term manure fertilization, but only co-localized with proteins in the no fertilization and chemical fertilization. To summarize, our findings highlight the importance of manure inputs in increasing formation of mineral-associated organic matters and thus potentially increasing soil carbon persistence.