The development of chromatographic media with superb adsorption capacity and large processing throughput is of great importance for highly efficient protein adsorption and separation, yet still faces a huge challenge. Herein, a new kind of butane tetracarboxylic acid (BTCA) functionalized ethylene vinyl alcohol (EVAL) nanofibrous membranes (BTCA@EVAL NFM)-based chromatographic media is fabricated, for the first time, by combining blend electrospinning technique with in-situ modification technology. The resulting BTCA@EVAL NFM possesses an enhanced equilibrium protein adsorption capability (716 mg g(-1)), a high saturated dynamic protein binding capacity (490 mg g(-1)), and a distinctive selectivity towards positively charged proteins, which are attributed to the synergistic effects of the hydrophilic EVAL nanofibrous matrix and the plentiful carboxyl groups introduced by BTCA. Besides, benefiting from its stable physical and chemical structures, the membrane also presents excellent acid and alkaline resistance as well as good reusability. Significantly, the BTCA@EVAL NFM can directly extract lysozyme from egg white with a relatively large capture capability of 353 mg g(-1), highlighting its superb potential practicability. We sincerely hope that this new design concept and the highly effective nanofiber-based chromatographic media can provide some guidance for the further development of bio-separation and purification. (C) 2019 Elsevier Inc. All rights reserved.