Enzyme immobilization provides improve mechanical characteristic of enzymes and stability to environmental alters. It also helps to enzymes maintain their activities for a long time and offers the possibility to use them again. With these unique features, they can reduce production costs in industrial applications. In this study, acetylcholinesterase (AChE) immobilized on Zn2+-doped PVA nanofibers via adsorption and cross-linking methods. Morphological structures of Zn2+-doped PVA nanofibers were characterized by Scanning Electron Microscopy (SEM) before and after immobilization. It was observed that the enzyme gained resistance against temperature and pH changes occurring in the reaction medium after immobilization process. In addition, immobilized Zn2+-doped PVA nanofibers retained of its activity approximately 75 % after 8 reuse and its activity decreased below 50 % after 12 reuse. As a result, AChE immobilized Zn2+-doped PVA nanofibers offers numerous advantages for various biotechnological applications, such as hydrolysis and determination of pesticides, use in the structure of reactors and electrodes, thanks to the unique stability and reusability features that AChE gains after immobilization.