Rhizopus oryzae lipase (ROL) was loaded on Fe3O4 superparamagnetic nanoparticles (MNPs) with electrostatic attractions and covalent bonding. The nano-biocatalysts were used in biodiesel production process to investigate the effect of enzyme covalent bonding on catalytic performance. For nano-biocatalysts preparation, ROL was loaded on pure MNPs and MNPs functionalized with 3-aminopropyl triethylenesilane (MNP-AP), electrostatically. The enzyme was also bonded to MNPs functionalized with 3-aminopropyl triethylenesilane-glutaraldehyde (MNP-AP-GA), covalently. The hydrolytic activity, kinetic parameters, esterification productivity, and reusability of ROL/MNP, ROL/MNP-AP, and ROL/MNP-AP-GA as the four nano-biocatalysts were investigated and compared. The morphology, structure and magnetic characteristics of the MNPs were evaluated using FESEM, FTIR, XRD and VSM. The components of products were analyzed using GC-MS technique. The covalent bonding increased enzyme loading on the support from 36.35 to 51.75 wt %. Transesterification reaction in the presence of ROL/MNP-AP-GA showed the highest conversion (69.8 wt %) among immobilized biocatalysts. Reusability analysis showed that the covalent bonding of enzyme (ROL/MNP-AP-GA) can prevent catalyst wasting, significantly. The conversion after five cycles of reuse for ROL/MNP-AP-GA was 2 times higher than ROL/MNP-AP and 3 times higher than ROL/MNP. The covalent bonding of lipase using relatively long chain coupler agents showed to be a reliable method for enhancing enzyme loading and productivity. (C) 2019 Elsevier Ltd. All rights reserved.