Agarose hydrogel was enriched by chitosan as an active substance for the interactions with dyes. Direct blue 1, Sirius red F3B, and Reactive blue 49 were chosen as representative dyes for the study of the effect of their interaction with chitosan on their diffusion in hydrogel. Effective diffusion coefficients were determined and compared with the value obtained for pure agarose hydrogel. Simultaneously, sorption experiments were realized. The sorption ability of enriched hydrogel was several times higher in comparison with pure agarose hydrogel. Determined diffusion coefficients decreased with the addition of chitosan. Their values included the effects of hydrogel pore structure and interactions between chitosan and dyes. Diffusion experiments were realized at pH 3, 7, and 11. The effect of pH on the diffusivity of dyes in pure agarose hydrogel was negligible. Effective diffusion coefficients obtained for hydrogels enriched by chitosan increased gradually with increasing pH value. Electrostatic interactions between amino group of chitosan and sulfonic group of dyes resulted in the formation of zones with a sharp boundary between coloured and transparent hydrogel (mainly at lower pH values). A concentration jump was observed at a given distance from the interface between hydrogel and the donor dye solution.