tA novel method using particle-packing technique to fabricate capillary electrophoresis (CE)-based immo-bilized enzyme reactor (IMER) was accomplished by utilizing perfusive silica single particles as the fritsand large-pore beads as the enzyme supports. The fabrication procedure is rapid and simple; the lengthand enzyme loading amount of the CE-IMERs could be easily adjusted. Performance and feasibility of theCE-IMERs were investigated using on-line trypsin digestion as the model enzyme reaction. High repro-ducible on-line enzyme assay was demonstrated with RSD less than 4.1% and 3.8% for peak area andmigration time of the substrate and product over 100 consecutive runs, respectively. The enzyme canstill maintain the activity for at least 10 days, indicating remarkably stability of the CE-IMERs. The CE-IMERs were successfully applied for accurate analysis of trypsin inhibition as well as on-line digestionof standard proteins (myoglobin and BSA). The present method provides a new interesting alternativeto open-tubular and monolithic CE-IMERs, thus expands the application of the CE technique for on-lineenzyme assay and analysis and characterization of peptides and proteins.