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Engineering 3D nanofibrous monolithic materials with a controllable porous channel structure and chemical activity is promising for the rapid and efficient separation of environmentally sensitive biomacromolecules such as RNA. Herein, freeze-drying method via regulating the viscosity of nanofiber dispersions is developed to fabricate nanofibrous sponges (NFSs) with 3D structures of microsphere, lamella, and honeycomb by combining poly(vinyl alcohol-co-ethylene) (PVA-co-PE) nanofibers with chitosan (CS) and polyethyleneimine (PEI) in dispersions. Among sponges, a honeycomb nanofibrous sponge (HNFS) possesses highly oriented open-cells and optimal performance, including water permeability of 7.431 × 10 3 L h m −2 at 1 psi, static saturated capacity of 887.6 mg g −1 , dynamic capacity of 763.2 mg g −1 at a flow rate of 1 mm min −1 , and mechanical robustness, superior to reported state-ofthe-art ion-exchange chromatography materials. Furthermore, HNFS-packed chromatographic, funnel, and centrifugal columns present exceptional performance and broad applicability in rapid-separation of RNA from model solution and diverse organisms without degradation. They are attributed to the highly oriented open-cell structure and high-density adsorption ligands on the surface of nanofibrous cell walls. This work provides a facile approach to design advanced NFSs with versatile physicochemical structure and outstanding performance.

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