The transport properties of agarose hydrogels enriched by humic acids were studied. Methylene blue, rhodamine 6G and Cu(II) ions were incorporated into hydrogel as diffusion probes, and then their release into water was monitored. Cu(II) ions as well as both the dyes studied in this work have high affinity to humic substances and their interactions strongly affected their diffusion in hydrogels. It was confirmed that humic acids retarded the transport of diffusion probes. Humic acids’ enrichment caused the decrease in the values of effective diffusion coefficients due to their complexation with diffusion probes. In general, the diffusion of dyes was more affected by the complexation with humic acids in comparison with Cu(II) ions. The effect of complexation was selective for the particular diffusion probe. The strongest effect was obtained for the diffusion of methylene blue. It was assumed that metal ions interacted preferentially with acidic functional groups. In contrast to Cu(II) ions, dyes can interact with acidic functional groups, and the condensed cyclic structures of the dye probes supported their interactions with the hydrophobic domains of humic substances.