Abstract: In this paper we studied one strain Aspergillus niger to produce alpha-L-rhamnosidase by fermentation, and isoquercitrin was prepared by curve enzyme with rutin as raw material. The Aspergillus niger was grown in the medium which containing rutin as an agent, and secretion alpha-L-rhamnosidase. The enzyme fermentation broth was precipitated by 70% ammonium sulfate, and the crude enzyme solution of alpha-L-rhamnosidase was obtained by dialysis. The enzyme activity was detected as 798 U/mL. The crude enzyme was then used to hydrolyze rutin into isoquercitrin. The molecular weight of the enzyme protein was 55 kDa by SDS-PAGE electrophoresis. The optimal temperature and pH of the enzyme were 55℃ and 5.5, respectively. The conversion rate of rutin reached 95% through in the optimum conditions described above. Using column chromatography method, isoquercetin purity could reach more than 99%. The enzyme also showed good thermal stability, and it can be used to convert rutin into quercetin, which has the advantages of high conversion rate, less by-products and high purity of the product. This study can provide an effective reference for the biotransformation of natural products.