The presence and identity of Cu-complexing ligands in soil solution strongly affects biogeochemistry, bioavailability and the fate of Cu in soils. In this study, we compared the influence of heavy metal pollution, vegetation and soil type on the amount and characterization of ligands able to form ternary complexes with Cu in soil solution. For separation and characterization, we applied immobilized metal ion affinity chromatography (IMAC) combined with fluorescence spectroscopy. All separated IMAC-fractions exhibited excitation-emission wavelengths of humic-like fluorescence (240–285/365–434 nm). Protein-type fluorescence (270–280/295–365 nm) and fluorescence at 330–340/375–385 nm were detected only in the retained fraction whereas carboxylate-type fluorescence (300–310/420–430 nm) was observed only in the non-retained fraction. These findings are in agreement with the behaviour of model ligands. The IMAC-retained ligands represented between 5 and 30% of dissolved organic carbon. The soil type and the vegetation had the largest influence on the quality and quantity of Cu ligands able to form ternary complexes. In the topsoil, the IMAC retained fraction was greater in soil without vegetation (16%) compared with soil with vegetation (12%). A larger amount (75%) of the protein-type ligands able to form ternary complexes with Cu was found in soil with vegetation compared with plant-free soil (69%). Metal pollution also affected the composition of the extracted ligands; the fraction with protein-type ligands decreased from 75% in unpolluted to 65% in the polluted topsoil. The results show that IMAC-retained ligands are related to the biological activity in soils.