There are different methods to modify polymer surfaces for biological applications. In this work we have introduced air-dielectric barrier discharge (DBD) plasma at atmospheric pressure as an economical and safe method for modifying the surface of electrospun chitosan/PEO (90110) nanofibers for acetylcholinesterase (AChE) immobilization. According to the contact angle measurement results, the nanofibers become highly hydrophilic when they are exposed to the DBD plasma for 6 min in compared to unmodified membrane. Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) results reveal hydroxyl, C=O and NH3+ polar groups increment after 6 min plasma treatment. Contact angle measurements and AIR-FTIR results are confirmed by X-ray photoelectron spectroscopy (XPS). AChE at pH 7.4 carries a negative charge and after immobilization on the surface of plasma-treated nanofibrous membrane attracts the NH3+ group and more enzyme activity is detected on the plasma-modified nanofibers for 6 min in compared to unmodified nanofibers. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) are used for the surface topography and morphology characterization. The results have proved that air-DBD plasma is a suitable method for chitosan/PEO nanofibrous membrane modification as a biodegradable and functionalized substrate for enzyme immobilization. (C) 2015 Published by Elsevier B.V.