alpha-l-Rhamnosidases have wide application in the field of biotechnology for derhamnosylation of many natural glycosides. In this study, an alpha-l-rhamnosidase-producing strain, Aspergillus niger CCTCC M 2018240, was isolated from decayed orange peels, and the gene encoding alpha-l-rhamnosidase was successfully expressed in Pichia pastoris GS115. Three-dimensional structure simulation indicates the enzyme is a member of glycoside hydrolase 78 family. The optimal recombinant strain GS115/pPIC9K-rha-14 exhibited an enzyme activity of 0.47 U/mL when cultured in shaking flasks, and the recombinant alpha-l-rhamnosidase hydrolyzed alpha-1,2 and alpha-1,6 glycosidic bonds in naringin and rutin, respectively, thus generating prunin and isoquercitrin, respectively. Through high density-induced fermentation based on a glycerol feeding strategy in a 3-L bioreactor, the enzyme activity reached 46.87 U/mL after 7 days of methanol incubation, which was approximately 99 times higher than that produced in shaking flasks. This process offers a simple and effective approach for the large-scale production of alpha-l-rhamnosidase.