Abstract: To study the coevolutionary of the functional residues and the variation of hydrolysis activity,the substrates binding residues simultaneous mutations of Geobacillus stearothermophilus thermostableβ-galactosidase were constructed.The results showed that the Y272 A/E351 R simultaneous mutant let a 3.67-fold increase in specific activity over the wild type enzyme and give a 2-fold increase over the single site mutation Y272 A.Increased Km value of Y272 A/E351 R mutant occurred with decreased affinity of lactose binding,while the mutant increased the catalytic efficiency of the wild type enzyme by 7.8-fold. This study revealed that the simultaneous evolution of the substrate binding residues had its advantage on substrate affinity,and was favorable to the evolution of catalytic activity.