► Facile preparation of Fe3O4–chitosan nanoparticles for enzyme immobilization. ► The characterizations of Fe3O4 and Fe3O4–chitosan nanoparticles were analyzed. ► Optimal immobilized activity 20U/g obtains at 2.14h, pH 6.37, and E/S ratio 0.73. ► Immobilized lipase could reuse 20 times while retaining 83% of its activity. ► The immobilized lipase shows better thermal, pH, and storage stability. Magnetic Fe3O4–chitosan nanoparticles are prepared by the coagulation of an aqueous solution of chitosan with Fe3O4 nanoparticles. The characterization of Fe3O4–chitosan is analyzed by FTIR, FESEM, and SQUID magnetometry. The Fe3O4–chitosan nanoparticles are used for the covalent immobilization of lipase from Candida rugosa using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) as coupling agents. The response surface methodology (RSM) was employed to search the optimal immobilization conditions and understand the significance of the factors affecting the immobilized lipase activity. Based on the ridge max analysis, the optimum immobilization conditions were immobilization time 2.14h, pH 6.37, and enzyme/support ratio 0.73 (w/w); the highest activity obtained was 20U/g Fe3O4–chitosan. After twenty repeated uses, the immobilized lipase retains over 83% of its original activity. The immobilized lipase shows better operational stability, including wider thermal and pH ranges, and remains stable after 13 days of storage at 25°C.