Non-stationary diffusion of two cationic dyes (Methylene Blue and Rhodamine 6G) was studied in hydrogels with different content of agarose and humic acids (HA). A simple spectrophotometrical method was utilized in the in situ measurement of dye concentration in the gel samples at different distances from the boundary. The effect of temperature, pH and ionic strength was investigated. The results confirmed the considerable partitioning of both dyes in agarose gels as well as the strong immobilization of dyes caused by their sorption on HA. The apparent diffusion coefficients of both dyes decreased with increasing solid content in gels. In the case of agarose gels without the addition of HA, this decrease was attributed to increased tortuosity of diffusion caused by denser agarose network. The apparent equilibrium constant of the sorption of dyes on HA in agarose/HA gels was calculated from their apparent diffusion coefficients. The value of the equilibrium constant increased with the content of HA in gel and, surprisingly, also with decreasing pH inside gel. (C) 2013 Elsevier Ltd. All rights reserved.