Progression of enzymes in lignocellulosic biomass is a crucial parameter in biorefinery processes, and it appears to be one of the limiting factors in optimizing lignocellulose degradation. In order to assay the importance of the chemical and structural features of the substrate matrix on enzyme mobility, we have designed bioinspired model assemblies of secondary plant cell walls, which have been used to measure the mobility of fluorescent probes while modifying different parameters (probe size, water content, polysaccharide concentration). The results were used to construct a model of probe mobility and to rank the parameters in order of importance. Water content and probe size were shown to have the greatest effect. Although these assemblies are simplified templates of the plant cell walls, our strategy paves the way for proposing new approaches for optimizing biomass saccharification, such as selecting enzymes with suitable properties.