α-L-rhamnosidases play a key role in the metabolism and biodegradation of dietary flavonoid glycosides. We have developed a novel microplate spectrophotometric method to rapidly evaluate the conversion rates and substrate selectivities of mesophilic α-L-rhamnosidases towards citrus flavanone diglycosides by combining with a high-active and thermophilic β-D-glucosidase based on UV-visible spectral differences between citrus flavanone diglycosides and the corresponding aglycones under alkaline conditions. Furthermore, catalytic activities and enzyme kinetics of four α-L-rhamnosidases from human gut bacteria on various dietary flavonoid glycosides with different glycosidic bonds from various subclasses have been explored by HPLC. The α-L-rhamnosidase BtRha78A specifically removed the rhamnose group from the flavones, flavanones and flavonols diglycosides with the α-1,6 glycosidic bonds. Moreover, BtRha78A displayed higher catalytic activities on the rutinose group at 7-OH of the aglycones than at 3-OH. HFM-RhaA preferred to catalyze the flavones, flavanones and dihydrochalcones diglycosides with the α-1,2 glycosidic linkages at the 7-OH. However, this enzyme also showed high catalytic activity on the flavonol diglycoside rutin with the α-1,6 glycosidic bonds at the 3-OH. HFM-RhaC exhibited certain hydrolytic abilities towards all flavonoid diglycosides, and displayed higher activities on the flavonoid diglycosides with the α-1,6 glycosidic bonds. HFM-Rha78 weakly hydrolyzed the flavones, flavanones and dihydrochalcones diglycosides with the α-1,2 glycosidic bonds, and the flavonols diglycosides with α-1,6 glycosidic bonds. All four α-L-rhamnosidases from human gut bacteria did not exhibit catalytic activity towards the flavonoid glycosides with the α-1 glycosidic bonds. It was revealed that the α-L-rhamnosidases from human gut bacteria possessed diverse substrate selectivity on dietary flavonoid diglycosides. The structural basis for the specificity of BtRha78A on the flavonoid diglycosides with α-1,6 glycosidic bonds and the preference of HFM-RhaA on the flavonoid diglycosides with α-1,2 glycosidic bonds have been analyzed by molecular docking.