Macromolecular crowding is an ubiquitous phenomenon in living cells that significantly affects the function of enzymes in vivo. However, this effect has not been paid much attention in the research of the immobilization of enzymes onto mesoporous silica. Herein, we report the combined effects of macromolecular crowding and surface hydrophobicity on the performance of an immobilized enzyme by accommodating lipase molecules into a series of mesoporous silicas with different amounts of inert poly(methacrylate) (PMA) covalently anchored inside the nanopores. The incorporation of the PMA polymer into the nanopores of mesoporous silica enables the fabrication of a crowded and hydrophobic microenvironment for the immobilized enzyme and the variation in polymer content facilitates an adjustment of the degree of crowding and surface properties of this environment. Based on this system, the catalytic features of immobilized lipase were investigated as a function of polymer content in nanopores and the results indicated that the catalytic efficiency, thermostability, and reusability of immobilized lipase could all be improved by taking advantage of the macromolecular crowding effect and surface hydrophobicity. These findings provide insight into the possible functions of the macromolecular crowding effect, which should be considered and integrated into the fabrication of suitable mesoporous silicas to improve enzyme immobilization.