The invention relates to application of an alpha-L-rhamnoside enzyme in the directional synthesis of isoquercitrin by the biological conversion of rutin. Because the crude enzyme preparation or the immobilized enzyme preparation of the alpha-L-rhamnoside enzyme is used for catalyzing and hydrolyzing the rutin and directionally and biologically synthesizing the isoquercitrin, the application has the advantages of wide source, easy preparation and low cost of catalysts, high stability, and high catalysis efficiency and specificity of the enzyme preparation, and is easy to store. Thus, the alpha-L-rhamnoside enzyme can greatly reduce the production cost of quercetin, the catalysis and conversion rate of the enzymes is high, and the products are single.