L-Asparaginase (L-ASNase) is a vital enzyme for medical treatment and food industry. Here, we assessed the use of Fe3O4@Mobil Composition of Matter No. 41 (MCM-41) magnetic nanoparticles as carrier matrix for L-ASNase immobilization. In addition, surface of Fe3O4@MCM-41 magnetic nanoparticles was functionalized with 3-mercaptopropyltrimethoxysilane (MPTMS) to enhance stability of L-ASNase. The chemical structure, thermal properties, magnetic profile and morphology of the thiol-functionalized Fe3O4@MCM-41 magnetic nanoparticles were characterized with Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), differential thermal analysis (DTA), differential scanning calorimetry (DSC), vibrating sample magnetometer (VSM), scanning electron microscope (SEM), energy dispersive X-ray (EDX) spectroscopy and zeta-potential measurement. L-ASNase was covalently immobilized onto the thiol-functionalized Fe3O4@MCM-41 magnetic nanoparticles. The properties of the immobilized enzyme, including optimum pH, temperature, kinetic parameters, thermal stability, reusability and storage stability were investigated and compared to free one. Immobilized enzyme was found to be stable over a wide range of pH and temperature range than free enzyme. The immobilized L-ASNase also showed higher thermal stability after 180 min incubation at 50 degrees C. The immobilized enzyme still retained 63% of its original activity after 16 times of reuse. The Km value for the immobilized enzyme was 1.15-fold lower than the free enzyme, which indicates increased affinity for the substrate. Additionally, the immobilized enzyme was active over 65% and 53% after 30 days of storage at 4 degrees C and room temperature (similar to 25 degrees C), respectively. Thereby, the results confirmed that thiol-functionalized Fe3O4@MCM-41 magnetic nanoparticles had high efficiency for L-ASNase immobilization and improved stability of L-ASNase.