Activity losing during the covalent immobilization of enzyme is a serious problem. Here we studied organic phase immobilization by using glucose oxidase (GOD) as a model. After lyophilized at optimum pH, GOD is covalently immobilized onto glutaraldhyde-activated chitosan microsphere carrier under the condition of water, 1, 4-dioxane, ether and ethanol separately. The special activities, enzyme characterization and kinetic parameters are determined. Results show that all of the organic phase immobilized GODs have higher special activities and larger Kcat than that of aqueous phase. Under the conditions of 0.1% of glutaraldehyde,1.6% moisture content with 80 mg of GOD added to per gram of carrier, 2.9-fold of the special activity and 3-fold of the effective activity recovery ratio were obtained, and 3-fold of the residue activity was demonstrated after 7 runs when compares1, 4-dioxane phase immobilized GOD with water phase immobilized one. In addition, kinetic study shows that 1,4-dioxane immobilized GOD (Kmapp=5.63 mmol/L, Vmax=1.70 μmol/(min·mg GOD), Kcat=0.304 s?1) was superior to water immobilized GOD (Kmapp=7.33 mmol/L, Vmax=1.02 μmol/(min·mg GOD), Kcat=0.221 s?1). All above indicated GOD immobilized in proper organic media presented a better activity with improved catalytic performance. Organic phase immobilization might be one of the ways to overcome the conformational denature of enzyme protein during covalent modification.