α-L-Rhamnosidase is an important biocatalyst for the synthesis of bioactive flavonoid compounds. However, its industrial application is often limited by low catalytic efficiency and insufficient thermostability. In this study, based on a previously obtained single-point mutants, multiple double mutants were constructed using a combined mutation strategy to further enhance enzyme performance. The best-performing mutant, R783A/R386A, was successfully identified. Its relative enzyme activity was 7.63 times that of the wild-type enzyme, and its thermostability was significantly improved, retaining 90.4% residual activity after incubation at 70 °C for 3 h. Kinetic analysis of the enzymatic reaction showed that the Km of R783A/R386A decreased by 73%, resulting in a 2.4-fold increase in the apparent specificity constant (kcat·Km-1) at low substrate concentrations; however, it should be noted that this increase in the ratio was accompanied by a 36% decrease in kcat, revealing a negative trade-off between enhanced affinity and reduced turnover number, indicating that the mutation primarily optimized the substrate binding step. In the synthesis of isoquercitrin using rutin as a substrate, R783A/R386A exhibited excellent catalytic performance, achieving a yield of 97%. Molecular mechanism analysis demonstrated that the R783A/R386A mutant markedly enhanced enzyme–substrate interactions and catalytic activity by optimizing the conformational flexibility of the catalytic binding pocket, which providing an important enzyme resource and a theoretical basis for the industrial application of α-L-rhamnosidase and the rational engineering of glycosidases.