The investigation on fabrication of Fe3O4-chitosan-pectinase nanobiocatalyst was performed by covalently binding the pectinase onto carboxyl group activated chitosan-coated magnetic nanoparticles (CMNPs). The morphological and size distribution analysis of the different magnetic nanoparticles (MNPs) was done using transmission electron microscopy (TEM), and the average diameter was 11.07 +/- 3.04, 11.55 +/- 3.16, and 11.59 +/- 3.16 nm for MNPs, CMNPs, and fabricated nanobiocatalyst, respectively, suggesting that there was no significant change in the size of MNPs after coating and binding. The characteristic peaks occurred at 2 theta of 30.39, 35.43, 43.37, 57.22, and 62.9, and their corresponding indices 220, 311, 400, 520, and 441 for different MNPs from the X-ray diffraction (XRD) studies confirmed the presence of Fe3O4 with the spinel structure, and there was no phase change even after coating and binding. The various required characteristic absorption peaks (575, 585, 1,563, 1,614, 1,651, and 1,653 cm(-1)) from Fourier transform infrared (FT-IR) spectroscopy confirmed the surface modifications and binding of pectinase onto the MNPs. At the weight ratio of about 19.8 x 10(-3) mg bound pectinase/mg activated CMNPs, the activity of fabricated nanobiocatalyst was found to be maximum. In order to monitor their improved activity, the pH, temperature, reusability, storage ability, and kinetic studies were established.