α-L-rhamnosidase [E.C. 3.2.1.40] is important in various industrial and biotechnological applications. However, limited knowledge of the structural features of its active site residues and their local geometric arrangements during substrate interaction hinders further application development. In this study, we examined functionally characterized microbial α-L-rhamnosidases. Despite considerable differences in their global structures, the local structures of the substrate-binding sites and key residues were highly conserved. Using the local structural motif, we characterized α-L-rhamnosidase genes from metagenomic samples of traditional fermentation starters. To comprehensively understand the distribution of α-L-rhamnosidases with this motif in the AlphaFold database, we screened 26,858 α-L-rhamnosidase structures. Our findings showed that only 5678 out of 26,858 structures contain the specific conserved motifs, emphasizing their potential significance in mining enzyme function. Moreover, the analysis of structural diversity among representative enzymes demonstrated variation in the number and types of domains within this enzyme family. Further investigation of representative α-L-rhamnosidase sequences with this structural motif confirmed the evolutionary constraints of 15 key residues, indicating strong selective pressures to maintain these elements essential for enzyme functionality. These residues were consistently present across ancestral sequences, underscoring their importance throughout the enzyme's evolutionary history. This study suggests that structure-guided approaches are valuable for discovering functional enzymes. Identifying conserved motif across diverse microbial taxa not only aids in predicting enzyme functionality but also offers opportunities for enzyme engineering and biotechnological applications.