Abstract: By using a compound method of adsorption, embedment and crosslink, a bifunctional enzyme produced by Providencia sp. JNB815 with both urease and urethanase activity was immobilized. With urea degrading activity as an indicator, the optimum immobilization conditions through single factor experiments were as follows:chitosan 4.0%, gelatin 2.0%, 0.4% of genipin and the crosslink was conducted at 30℃ with concussion for 6h. Then the crosslinked microspheres were used to immobilize the enzyme for 8h. The optimum pH was 4.5 and 4.0, and the optimum temperature was 60℃ and 40℃, respectly when immobilized enzyme catalyzed urethane and urea. The immobilized enzyme activity to EC maintained stability when the temperature was in the range of 20⁶0℃ and pH 5.0 to 6.5. The immobilized enzyme activity to urea maintained stability when the temperature was in the range of 20⁵0℃ and pH 4.0 to 6.5.It retained 80% and 30% of the original catalytic avtivity to urea and EC, respectively after being used 10 times. Under the condition of 32℃ and100r/min, 0.04U urease was added to per mL rice wine. After being processed for 20h, the removing rate of urea in wine reached 93.03%. Under the same condition, the removing rate of EC in wine was 56.60% after20h, when the amount of urethanase added was 0.17U per mL wine. In addition, the treatment with immobilized enzyme showed neglectable effect on the detected flavor substances in the wine.