Monoclonal antibodies (mAbs) are one of the most promising classes of therapeutic protein biopharmaceuticals. However, the complexity of mAbs poses a daunting analytical challenge for heterogeneity characterization of mAbs. In this study, inspired by blood coagulation, we adopted a fibrin coating as a novel stationary phase in open tubular (OT) column for the separation of the mAbs variants by capillary electrochromatography. The fibrin coating was prepared by in situ polymerization of fibrin in the presence of thrombin as a catalyst inside a fused-silica capillary. Scanning electron microscopy and electroosmotic flow measurement were carried out to characterize the fibrin coated OT columns. The average thickness of the fibrin coating was about 1.13 μm. And the EOF of the column was pH-dependent. The electrochromatographic performance of the prepared columns was evaluated by characterization of the variants of three mAbs (cetuximab, trastuzumab and rituximab). The columns demonstrated good repeatability with the run-to-run, day-to-day and column-to-column relative standard deviations of migration times less than 2.42%. The study highlighted the potential of adsorbed proteins as stationary phases for the separation of mAbs variants. Furthermore, the study provided a new platform for characterization of heterogeneity of mAbs in pharmaceutical industry.