Previously, we developed a novel separation technique, namely, supported molecular matrix electrophoresis (SMME), which separates mucins on a PVDF membrane that impregnated with a hydrophilic polymer (such as polyvinyl alcohol), so it has the characteristics that are compatible with glycan analysis of the separated bands. Here, we describe the first instance of the application of SMME to mouse sera fractionation and demonstrate their differences from the pooled human sera fractionation by SMME. Furthermore, we have developed a fixation method for the lectin blotting of SMME-separated glycoproteins by immersing the SMME membranes into acetone solvent followed by heating. It showed that the amount of protein samples required for SMME were reduced more than 4-fold than that of the process of SDS-PAGE. We applied these techniques for the detection of glycosylation patterns of serum proteins from Fut8+/+ and Fut8-/- mice, further analyzed N-linked and O-linked glycans from the separated γ-bands by mass spectrometry, and demonstrated that there are α2,8-sialylated O-glycans contained in mouse sera glycoproteins. SMME can provide simple, rapid sera fractionation, glycan profiling differences between the bands of two samples and a new insight into the underlying mechanism that responsible for related diseases. SIGNIFICANCE: We describe that the first application of SMME can separate mouse serum proteins into six bands and identify the major protein components of each fraction in mouse serum separated by SMME. Furthermore, we successfully developed a fixation method for lectin blotting of SMME-separated glycoproteins and applied to the detection of glycosylation patterns of serum glycoproteins from Fut8+/+ and Fut8-/- mice, also, the method is promising for detecting glycan profiling differences between two samples in both research and clinical settings.