Significance It is recognized that an optimized mixture of endo- and exo-glucanases, beta-glucosidases, and accessory enzymes need to work cooperatively to result in effective cellulose hydrolysis. However, a key factor that impacts hydrolysis is the effectiveness of enzyme access to the cellulose. A quantitative method based on fluorescence-tagged CBMs and CLSM was developed to assess the cellulose structure, quantify its accessibility, and elucidate how this might influence the efficiency of cellulose hydrolysis. This work provides insights into the substrate–enzyme interactions that occur during enzyme-mediated hydrolysis of cellulose.