Abstract: : β-Glucosidase can hydrolyze aryl-glucoside and holds significant application value in the fields of food and biotransformation. To investigate the influence of substrate side-chain groups on the activity of β-glucosidase, the β-D-Glu from Aspergillus niger was cloned and expressed. Through in silico saturation mutagenesis followed by experimental validation, key amino acid was identified within 4 Å of the β-D-Glu binding pocket, molecular docking and molecular dynamics simulations were employed to analyze the structure-activity relationship between the enzyme and substrate. β-D-Glu exhibited significant differences in activity toward structurally similar monocyclic aryl-glycoside substrates with varying side chains. Specifically, the enzymatic activities toward salicin, arbutin, and pNPG were measured at 44.72, 103.97, and 839.02 IU/mL, respectively. Through in silico saturation mutagenesis followed by experimental validation, residue R98 was identified as a key amino acid within 4 Å of the β-D-Glu binding pocket. Mutation at this site exerted substrate-specific effects on catalytic activity towards different substrates. Compared to wild-type enzyme, R98F mutation increased the relative activity towards salicin to 266.08%, while R98I and R98Q mutations decreased the relative activities to 37.62% and 35.05%, respectively. All three mutants showed decreased relative activities towards arbutin, but the relative activities towards pNPG remained almost unchanged. Specifically, the R98F mutant exhibited significantly enhanced activity towards salicin, while its activity towards arbutin and pNPG decreased by 16.44% and 7.54%, respectively. Molecular docking and molecular dynamics simulations revealed that differences in the polarity and electron-donating capacity of the side-chain groups in monocyclic aryl-glucoside substrates lead to distinct binding modes with the enzyme. The R98F mutation modulates both the conformational flexibility at the binding pocket entrance and the binding affinity between catalytic key residues and substrates. It is speculated that the mutation specifically enhances the hydrolytic activity toward salicin by improving both glycosidic bond cleavage efficiency and reaction rate. The results of this study help to reveal the hydrolysis characteristics of β-glucosidase towards different aryl- glucoside substrates and provide a scientific basis for the design and modification of this enzyme.