The active-site architecture of cellulase is a functional area composed of multiple amino acid residues which can bind and catalyze substrates. Aromatic residues, such as tryptophan, play important roles in the function of this region. In this study, the dynamic of binding process between tryptophan in active-site architecture of cellulase ChCel5A and substrate was quantitatively analyzed using fluorescence spectrum. The substrate concentration range when tryptophan can specifically bind the substrate was determined and the change of binding constant caused by a single amino acid mutation in active-site architecture of ChCel5A was measured through quantitative analysis of tryptophan fluorescence quenching. Compared with the measurement of kinetic parameters, fluorescence spectroscopy could accurately characterize the binding affinity between cellulase and substrate, as well as the change of kinetic parameter caused by a single residue mutation. Besides, when using pNPC as the ligand, the binding constant could be overestimated for 20-100 times due to the increase of strong electrophilic groups. Fluorescent spectrometry can measure the kinetic parameter of cellulase-substrate binding process in a sensitive and rapid way, providing a new perspective for the quantitatively analyzing the interaction between proteins and substrates.