Abstract Tannic-acid-templated large pore mesoporous silica nanoparticle (LMSN) was synthesized using one-pot procedure and the external surface was further modified with APTES (LMSN-N) to immobilize trypsin. After modification, trypsin was difficult to be immobilized on the outer surface of the LMSN due to the electrostatic repulsion. Naturally, study of the trypsin immobilized in the interior pores could be implemented solely. Meanwhile, the activity of immobilized trypsin was optimized by response surface method (RSM), and the ANOVA was employed to explore the interaction between independent variables. The maximum enzyme activity (12.28 U/mg) was obtained under a certain condition that the quality of T-LMSN-N-272 was 153 μg, the pH was 7.9, and the temperature was 40.4 °C. Simultaneously, the immobilized enzyme was proved to possess better thermal stability, storage stability and recycling performance than free enzyme.