The soil-water interfaces (SWI) in soil pores are hotspots for organic matter (OM) transformation. However, due to the heterogeneous and opaque nature of soil microenvironment, direct and continuous tracing of interfacial reactions, such as OM transformations and formation of organo-mineral associations, are rare. To investigate these processes, a new soil microarray technology (SoilChips) was developed and used. Homogeneous 800-μm-diameter SoilChips were constructed by depositing a dispersed Oxisol A horizon suspension on a patterned glass. Dissolved organic matter from the original soil was added on the SoilChips to mimic SWI processes. The effects of ammonium fertilization (90 mg N kg−1 soil) on chemical composition of SWIs were evaluated via X-ray photoelectron spectroscopy. Over 21 days, ammonium addition increased OM coatings at SWIs and modified the OM chemical structure with more alcoholic- and carboxylic-C compared to the unfertilized control. Molecular modeling of OM composition at SWIs showed that N fertilization mainly facilitated the microbial production of glucans. We demonstrated that N availability modifies the specific OM molecular processing and its immobilization on SWIs, thereby providing a direct insight into biogeochemical transformation of OM at micro-scale. © 2019, The Author(s).