Dissolved organic matter (DOM) in soil solution represents a complex mixture of organic molecules and plays a central role in carbon and nitrogen cycling in plant–microbial–soil systems. We tested whether excitation–emission matrix (EEM) fluorescence spectroscopy can be used to characterize DOM and support previous findings that the majority of DOM is of high molecular weight (MW). EEM fluorescence spectroscopy was used in conjunction with MW fractionation to characterize DOM in soil solution from a grassland soil land management gradient in North Wales, UK. Data analysis suggested that three distinct fluorescence components could be separated and identified from the EEM data. These components were identified as being of humic-like or fulvic-like origin. Contrary to expectations, the majority of the fluorescence signal occurred in the small MW (<1 kDa) fraction, although differences between soils from the differently managed grasslands were more apparent in larger MW fractions. We conclude that following further characterization of the chemical composition of the fluorophores, EEM has potential as a sensitive technique for characterizing the small MW phenolic fraction of DOM in soils.