A Structure‐Guided Multistrategy Design Enhances Thermostability of an α‐L‐Rhamnosidase for Efficient Biotransformation

α‐L‐Rhamnosidase is a key biocatalyst for natural product biotransformation, but its poor thermostability limits industrial applications. To address this challenge, this study developed a structure‐guided multiplatform rational design strategy and applied it to a Bifidobacterium α‐L‐rhamnosidase (BcRha), identifying T384P as a key thermostable mutant. T384P exhibited a half‐life of 353.3 min at 60 °C (3.3‐fold that of the wild type, WT) and 81.8 min at 65 °C (4.4‐fold), together with a 4.3 °C increase in melting temperature. Mechanistic analyses indicated that T384P enhanced thermostability by strengthening long‐range dynamic coupling, establishing a multipoint polar locking network, and reducing global conformational fluctuations. Notably, combination mutants failed to exhibit synergistic enhancement, possibly due to coupling interference between distinct dynamic modules. Under high substrate loading (30 g/L rutin), T384P achieved an 18.8% higher isoquercitrin yield than WT at 60 °C and retained substantial activity at 65 °C, approximately twice that of WT. This study provides an efficient rational design strategy for improving α‐L‐rhamnosidase thermostability, reveals a possible mechanistic basis for nonadditive mutation effects, and offers a promising biocatalyst for high‐temperature flavonoid biotransformation.