Fe3O4 nanoparticles (NPs) were synthesized by co-precipitating Fe2+ and Fe3+ in an ammonia solution. Fe3O4 NPs functionalized with tannic acid were prepared. After functionalization process, trypsin enzyme was immobilized on these Fe3O4 NPs. The influence of pH, temperature, thermal stability, storage time stability and reusability on non-covalent immobilization was studied. The properties of Fe3O4 and its modified forms were examined by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA), UV-vis spectrometer (UV) and X-ray diffraction (XRD), magnetization and zeta potential measurements. The immobilized enzyme was slightly more stable than the free enzyme at 45°C. According to the results, the activity of immobilized trypsin was preserved 55% at 45°C after 2 h and 90% after 120 days storage. In addition, the activity of the immobilized trypsin was preserved 40% of its initial activity after eight times of successive reuse.