The study is published in the journal Agronomy. Despite the essential roles of soil dissolved organic matter (DOM) and soil microbes in agro-ecosystems, we still have a limited understanding of the extent by which they are impacted by agronomic strategies in ecological and conventional farming. Using three-dimensional fluorescence excitation–emission matrices (3D-EEM) and high-throughput microbial sequencing, the characteristics of soil DOM and microbiota under realistic field conditions were estimated in the farming soils with long-term ecological (EM) and conventional management (CM). Specifically, the role of hedgerows in the ecologically managed land (EMH) was assessed. The total fluorescent intensity of soil DOM in the EMH system was significantly higher than the values in CM and EM systems. Additionally, the five normalized excitation–emission area volumes from regional integration analysis increased in the order CM < EM < EMH. In comparison with CM and EM soils, the hedgerow significantly increased the evenness of the bacterial communities in the EMH system, whereas no differences were found for the alpha-diversity of eukaryotic communities. The composition of soil microbiota was significantly distinct among the three farming systems, with a hedgerow-specific effect on bacterial community and a management-specific effect on eukarya. The predicted functional profiles indicated that the hedgerow showed a higher contribution to the dissimilarity of bacterial functions. Furthermore, the distinction of the soil microbiota was modulated by the soil DOM composition and significantly positive correlations between the microbiota involved in nutrient cycling and soil DOM were observed. The findings in this work strengthen our understanding of the different responses of bacterial and eukaryotic communities under the long-term ecological management and highlight the beneficial roles of hedgerows in increasing organic matter and modulating community assembly.