Supported molecular matrix electrophoresis (SMME), in which a hydrophilic polymer such as PVA serves as a support within a porous PVDF membrane, was recently developed. This method is similar to cellulose acetate membrane electrophoresis but differs in the compatibility to glycan analysis of the separated bands. In this report, we describe the first instance of the application of SMME to human serum fractionation, and demonstrate the differences with serum fractionation by cellulose acetate membrane electrophoresis. The SMME membrane exhibited almost no EOF during electrophoresis, unlike the cellulose acetate membrane, but afforded comparative results for serum fractionation. The visualization of each fraction was achieved by conventional staining with dye such as Direct Blue‐71, and objective quantification was obtained by densitometry after inducing membrane transparency with 1‐nonene. Immunostaining was also achieved. Moreover, mass spectrometric analysis of both N‐linked and O‐linked glycans from the separated bands was demonstrated. Serum fractionation and glycan profiling of each fraction using SMME will enable novel insights into the relationships between various glycosylation profiles and disease states.