Murraya koenigii leaf extract mediated cysteine-functionalized magnetite nanoparticles (Cys-FeO NPs) were synthesized by the precipitation method. Cys-FeO NPs were activated using glutaraldehyde cross-linker (Glu-Cys-FeO NPs), and were used to immobilize xylose reductase (XR-FeO NPs). The effects of immobilization process parameters such as concentration of NPs, enzyme and cross-linking time were examined. The physicochemical properties of free XR, NPs and XR-FeO NPs were investigated. Results showed that Cys-FeO NPs have cubic spinel phase structures with spherical shape and sizes ranging from 90 to 100 nm. The successful binding of XR on functionalized NPs was confirmed through functional and structural studies. Minor structural rearrangements were observed in XR-FeO NPs compared to free XR enzyme. Under optimized conditions, the residual activity of Free XR and immobilized enzyme was found to be 80 and 85% respectively. Kinetic constant (Km) of free XR depicted that the immobilized enzyme had an increased binding affinity for the substrate. © 2019 Elsevier B.V.