Abstract A novel protein sample pretreatment method based on ampholine immobilized polymer microsphere ([email protected]) was developed for the fractionation of intact proteins prior to protein digestion and peptide analysis to reduce the dynamic range of human plasma proteome. After incubation with our prepared [email protected], the captured plasma proteins were successively desorbed by 2M NaCl, 100mM glycine-hydrochloric acid, and 30% (v/v) acetonitrile with 0.1% (v/v) trifluoroacetic acid. The SDS-PAGE results showed the protein dynamic range in such three fractions was obviously reduced as compared with the native plasma. On-particle digestion was ultimately performed to release all proteins retained on [email protected] Followed by MuPIT analysis, the number of identified proteins in plasma was improved by 75% after [email protected] treatment. Furthermore, the spectral count of 9 high abundance proteins was decreased by 37.6–97.2%, and the identified low abundance protein (<100ngmL−1) number was increased from 4 to 17. These results demonstrated that the fractionation by [email protected] could efficiently decrease the protein dynamic range in abundance, beneficial to achieve the deep coverage identification of human plasma proteome.