Abstract In situ steady state fluorescence (ssF) measure- ment technique was applied to investigation of pyrene la molecules diffusion in and out of agarose gels. Gel samples with four different concentration of agarose were prepared. PVA-Py was syn by "click" chemistry method and dissolved in water thesized to use in diffusion experiments. The results were analyzed by using Fickian type and it found that and desorption processes of PvA-Py molecules in and out of agarose gel have two distinct regions for short and long diffusion times. Sorption and desorption coeffi cients were measured and it was seen that the diffusion rates were much larger at short times and at lower agarose concentrations.