Affinity membrane chromatography is of great importance for the separation and purification of proteins. Electrospun nanofibrous mats decorated with the glycosylated surfaces, combining the merits of large surface area and high porosity as well as good specificity with lectin, are regarded as superior affinity membranes. We report a simple and effective approach to acquire a new kind of glycosylated nanofibrous membrane (GNM). Poly(acrylonitrile-co-hydroxyethyl methacrylate) (PAHM) was electrospun into nanofibrous mat with an average fiber diameter of 200 nm and glucose ligands were bound on the nanofiber surface through a reaction between glucose pentaacetate and the hydroxyl groups of PAHM. Interactions between the GNM surfaces and proteins were studied by confocal laser scanning microscopy. Static and dynamic protein adsorptions were also evaluated. Results indicates that the GNM selectively recognizes lectin concanavalin A (Con A) while shows almost no affinity binding with another lectin peanut agglutinin (PNA). It also shows strong multivalent and reversible binding capability as well as high adsorption capacity to Con A. We suggest that the GNM is promising in affinity chromatography for the separation and purification of lectins. (C) 2010 Elsevier B.V. All rights reserved.