Nanofibrous polyacrylonitrile membranes (PANNFM) were obtained by electrospinning and then prepared for immobilizing acetylcholinesterase (AChE). Initially, the chemical modification of PANNFM with ethylenediamine produced reactive groups to overcome their inertness and hydrophobicity. The natural polymer, chitosan, was then tethered on the nanofibrous membranes to improve their biocompatibility. Scanning electron microscopy (SEM) and cross-section SEM were used to determine morphological and porosity changes of the membranes. The immobilized AChE had greater relative activity as well as thermal and storage stability compared to the free enzyme. The bound AChE showed excellent reusability. Chitosan-modified PANNFM was shown to be a suitable strategy for facile immobilization of AChE to produce a promising system that effectively supports biocatalysts.