During lipase-mediated biodiesel production, by-product glycerol adsorbing on immobilized lipase is a common trouble that hinders enzymatic catalytic activity in biodiesel production process. In this work, we built a hydrophobic pore space in macroporous ZIF-8 (named as M-ZIF-8) to accommodate lipase so that the generated glycerol would be hard to be adsorbed in such hydrophobic environment. The performance of the immobilized lipase in biodiesel production as well as its characteristics for glycerol adsorption were systematically studied. The PDMS (polydimethylsiloxane) CVD (chemical vapor deposition) method was utilized to get hydrophobic M-ZIF-8-PDMS with hydrophobic macropore space and then ANL ( lipase) was immobilized on M-ZIF-8 and M-ZIF-8-PDMS by diffusion into the macropores.ANL@M-ZIF-8-PDMS presented higher enzymatic activity recovery and better biodiesel production catalytic performance compared to ANL@M-ZIF-8. Further study revealed that less glycerol adsorption was observed through the hydrophobic modification, which may attribute to the improved immobilized lipase performance during biodiesel production and ANL@M-ZIF-8-PDMS remained more than 96% activity after five cycles' reuse. Through secondary structure and kinetic parameters' analysis, we found that ANL@M-ZIF-8-PDMS had lower extent of protein aggregation and twice catalytic efficiency (/) than ANL@M-ZIF-8.Hydrophobic pore space constituted in macroporous ZIF-8 for lipase immobilization greatly improved lipase catalytic performance in biodiesel preparation. The hydrophobic modification time showed negligible influence on the reusability of the immobilized lipase. This work broadened the prospect of immobilization of enzyme on MOFs with some inspiration.