Magnetic nanoparticles were coated with polyvinyl alcohol and activated with glutaraldehyde for trypsin immobilization. The prepared magnetic nanoparticles were characterized by transmission electron microscopy, fourier transform infrared spectroscopy, thermal gravimetric analysis, zeta potential meter and vibrating sample magnetometer. Free and immobilized trypsin showed optimum activity at pH 6.0, 30 degrees C and pH 7.0, 40 degrees C, respectively. Immobilized trypsin was more stable than the free enzyme at 40 degrees C. After immobilization, K-m of the immobilized trypsin increased, however, V-max value was almost the same with free trypsin. According to the results, the immobilized trypsin retained 50 % of its initial activity, whereas free trypsin retained 19 % of its initial activity after 12-days at 4 degrees C. Immobilized trypsin sustained 56 % of its initial activity after eight times of successive reuse. The performance of the immobilized trypsin was evaluated by digestion of cytochrome c. The peptide fragments in digest solution were determined by using MALDI-TOF mass spectrometry. Immobilized trypsin showed effective proteolytic activity in shorter time (15 min) than free trypsin (24h). Hence, immobilized trypsin on the polyvinyl alcohol coated magnetic nanoparticles could be promising biocatalyst for large-scale proteomics studies and practical applications. (C) 2020 Elsevier B.V. All rights reserved.