Nanofibers have a great potential for enzyme immobilization application due to their large surface area to volume ratio besides their porous structure. In this work, we produce polymethyl methacrylate (PMMA) nanofibers via electrospinning method in dimethylformamide (DMF) as solvent. Thereafter, we employ a chemical method on final PMMA nanofiberous web to covalently immobilize acetylcholinesterase (AChE) enzyme on membrane surface. Morphology and tensile properties of nanofibers are studied as first steps of characterization to make sure of obtaining a properly stable membrane for enzyme carrying application. Thereafter, the stability and activity of immobilized enzymes as two main characteristic parameters are tested and reported for different applications such as biosensor manufacturing.