In this communication, fenugreek β-amylase was immobilized onto functionalized tungsten disulfide nanoparticles through cross-linker glutaraldehyde and successful immobilization was confirmed by SEM, AFM and FTIR spectroscopy. To make the process economical and efficient, optimization of independent variables was carried out using Box-Behnken design of response surface methodology. Approximately similar predicted (85.6%) and experimental (84.2%) immobilization efficiency revealed that the model is suitable for design of space. Optimum temperature was calculated to be 60 °C. After immobilization, an increased Km (2.12 times) and a decreased Vmax (0.58 times), indicated inaccessibility of active site residues to the substrate. The immobilized enzyme retained 77% relative activity after 10 uses whereas 40% residual activity was obtained after 120 days. An increased half-life with concomitantly decreased kinetic rate constant revealed that the immobilized enzyme is more stable at a higher temperature and the process followed first-order kinetics (R2 > 0.93). The limit of detection for maltose and sucrose fluorescence biosensor was found to be 0.052 and 0.096 mM, respectively. Thermodynamic parameters such as changes in Gibbs free energy (ΔG < 0), enthalpy (ΔH > 0) and entropy (ΔS >0) revealed that the process is spontaneous and endothermic, driven by hydrophobic interactions. Thermo-stability data at higher temperature for the immobilized enzyme makes it a suitable candidate for industrial applications in the production of maltose in food and pharmaceutical industries. Furthermore, fluorescence biosensor could be used to detect and quantify maltose and sucrose to maintain the quality of industrial products. © 2019 Elsevier B.V.