Icariin, the most effective bioactive component in Epimedium, is also the index component of Epimedium quality control in Pharmacopoeia. It was a very attractive approach for bioconversion from epimedin C to icariin. However, its potential was impeded by poor stability and non-recyclable properties of free enzymes. In this study, we have fused the linker (4LP) to α-L-rhamnosidase BtRha and successfully prepared the immobilized enzyme (incubated [email protected]) to produce icariin from epimedin C. The activity recovery of [email protected] was 79.6%, and enzyme activity was 209.8 U/g, which was 1.75-fold and 1.6-fold higher than that of immobilized BtRha ([email protected]), respectively. The optimal reaction temperature and pH of [email protected] was 55°C and 6.5, respectively. The thermal stability of immobilized enzyme was significantly improved by incubation in phosphate buffer containing 20% glycerol and 10% fructose. The kcat/Km value of incubated [email protected] was 7.98×105 s-1M-1, which increased by 8% compared with free BtRha. Finally, under suitable conditions, 1 g/L epimedin C was transformed into icariin with icariin yield 75.1%, and the relative conversion rate retained 74.9% after reused 13 cycles. This experiment provides a new idea for one-step purification and immobilization of α-L-rhamnosidase for direct biotransformation from epimedin C to icariin, which will have great prospects in food and pharmaceutical production.