The enzyme molecule is natural effective biological catalyst. The enzyme - substrate (ES) complex forming in the first step imparts the catalytic efficiency of enzyme. Specifically, multiple amino acid residues composed the active site of enzyme, which created a precisely organized architecture for substrate recognition, binding and catalytic process. Therefore, the function of amino acid residues in active-site architecture has always been the research hotspot in protein engineering. The enzymes in GH11 and GH12 families have small molecular mass and large range of enzymology properties, which are good candidates to study the binding affinity of enzymes. In this study, the catalytic activity and band mobility of different mutants in the active-site architecture of TrCel12A and TlXynA could be rapidly demonstrated by affinity electrophoresis. The substrate binding affinity can be quantitatively characterized by quantitative regression analysis of the relative mobility of proteins at different substrate concentrations. At the same time, isothermal titration calorimetry and fluorescence spectroscopy were also used to determine the binding affinity of different mutants. For the mutants of TrCel12A, the parameters K_b measured by affinity electrophoresis were significantly related with those determined by isothermal titration calorimetry (R² = 0.82) and fluorescence spectroscopy (R² = 0.83). Therefore, the magnitude of their affinities was TrCel12A-E200Q>W22YE200Q>W22FE200Q>W22HE200Q>W22AE200Q, respectively. Furthermore, the substrate binding affinities of the variants in TlXynA were also characterized using affinity electrophoresis. The results showed that there was a good correlation with that measured by fluorescence spectrometry (R² = 0.84). These results indicate that the parameters K_b measured by affinity electrophoresis can characterize the binding ability influenced by amino acid single-point mutation in active-site architecture of glucoside hydrolase. Therefore, affinity electrophoresis was effective and can be used as routine screening technology in biochemical laboratory to detect the change of series mutants binding affinity in mutant library.