α-L-rhamnosidase produced by high density fermentation was immobilized into matrix consisting of polyvinyl alcohol and sodium alginate with glutaraldehyde as crosslinker. In the 5 L bioreactor, the activity of immobilized enzyme achieved 2 766 U/g and its biomass reached 228 g /L after 96 h cultivation. The enzyme activity recovery of 32.88% was obtained and the activity of immobilized enzyme was 20 U/g. The optimal temperature was 35-50 ℃ and the optimal reaction pH at about 4.0 for immobilized enzyme. The residual enzyme activity could retain more than 98.22% after 6 batches of recycling use. These results suggested that the high density fermentation could effectively enhance enzyme production, and the immobilization technology could improve enzyme recycling rate, which would offer a basis for large-scale production of isoquercitrin.