MALDI-TOF MS is being widely applied to analyze serum samples for the diagnosis of human diseases and for the identification of potential biomarkers. However, different types of magnetic beads afford different purification capability for low abundance proteins and peptides in body fluids. The aim of our study was to establish a proteome fractionation technique for choosing magnetic beads for proteomic analysis in breast cancer research. We used three different kinds of magnetic beads (MB-HIC C8, MB-IMAC-Cu and MB-WCX) to pre-analyze 28 breast cancer patients and 24 healthy control serum samples for proteomic profiling by MALDI-TOF MS analysis. The serum samples purified by MB-WCX group provided the best proteomic pattern .These samples had the most average peak numbers, the highest peak intensities, and the best capturing ability of low abundance proteins or peptides in serum samples. Therefore, we chose MB-WCX as our best magnetic beads to pre-analyze large-scale cancer and healthy control samples. The application of the standardized, pre-analytical serum samples with right type of magnetic bead-based fractionation will decrease variability of proteome patterns in human serum assessed by MALDI-TOF MS.