Enzyme-immobilized nanoparticles that are both catalysis effective and recyclable would have wide applications ranging from bioengineering and food industry to environmental fields; however, creating such materials has proven extremely challenging. Herein, we present a scalable methodology to create Candida rugosa lipase-immobilized magnetic nanoparticles (L-MNPs) by the combination of nonionic reverse micelle method and Fe3O4 nanoparticles. Our approach causes the naturally abundant and sustainable Candida rugose lipase to ordered-assemble into nanoparticles with high catalytic activity and durability. The resultant L-MNPs exhibit the integrated properties of high porosity, large surface area, fractal dimension, robust enzymatic activity, good durability, and high magnetic saturation (59 emu g-1), which can effectively catalyze pentyl valerate esterification and be easily separated by an external magnet in 60 second. The fabrication of such fascinating L-MNPs may provide new insights for developing functional enzyme-immobilized materials towards various applications.