In this study, an easy and efficiency protein digestion method called continuous microwave-assisted protein digestion (cMAED) with immobilized enzyme was developed and applied for proteome analysis by LC-MSn. Continuous microwave power outputting was specially designed and applied. Trypsin and bromelain were immobilized onto magnetic micropheres. To evaluate the method of cMAED, bovine serum albumin (BSA) and protein extracted from ginkgo nuts were used as model and real protein sample to verify the digestion efficiency of cMAED. Several conditions including continuous microwave power, the ratio of immobilized trypsin/BSA were optimized according to the analysis of peptide fragments by Tricine SDS-PAGE and LC-MSn. Subsequently, the ginkgo protein was digested with the protocols of cMAED, MAED and conventional heating enzymatic digestion (HED) respectively and the LC-MSn profiles of the hydrolysate was Compared. Results showed that cMAED combined with immobilized enzyme was a fast and efficient digestion method for protein digestion and microwave power tentatively affected the peptide producing. The cMAED method will be expanded for large-scale preparation of bioactive peptides and peptide analysis in biological and clinical research. (C) 2014 Elsevier Inc. All rights reserved.