This article describes a new way to immobilize an enzyme by enzymatic hydrogelation to facilitate catalysis in the organic solvent to attain high activity and stability. A self-immobilized acid phosphatase (AP) in a molecular hydrogel created by the enzymatic hydrogelation had shown higher activities in several organic solvents than those of free AP in the same solvents. The high activities of AP(gel) in organic solvents are mainly due to the cooperative effect of the amphiphilic nanofibers in the hydrogel and the phase transfer between the organic solvent and the water in the hydrogel. This approach provides a useful method to immobilize enzymes for biotransformation in organic solvents.