Sulfonated poly(ether ether ketone)-based membranes containing amphoteric functionalized nanofibrous network (SP@f) with the three-dimensional interlaced structure were developed, and the acid-base interaction between SP@f and SPEEK constructs highly conductive proton channel and compatible interfaces. The large specific surface area and aspect ratio of nanofibers afford an orientated ion transmission channel along the fiber axis, bringing a high concentration of proton carriers and abundant hydrogen bonds to transfer proton. The S/SP@f-10 composite membrane displays superior proton conductivity (37.8 mS cm −1 ) to pristine SPEEK (16.4 mS cm −1 ) and Nafion 212 (28.0 mS cm −1 ). Owing to the interconnected network structure of SP@f and the acid-base interactions, ion selectivity and chemical stability of the S/SP@f-10 composite membranes improve seriously. VRFB single cell with S/SP@f-10 membrane shows better voltage efficiencies (VE: 93.0–74.0%) and energy efficiencies (EE: 87.5–73.0%) than that of pristine SPEEK (VE: 86.0–60.2%, EE: 82.1–58.0%) and Nafion 212 membrane (VE: 88.0–67.0%, EE: 80.0–63.9%) at 60–200 mA cm −2 . Meanwhile, S/SP@f-10 maintains a stable EE value of 80.0%, and no obvious decay after 500 cycles at 150 mA cm −2 appears, demonstrating its outstanding structural stability and durability. This opens a facile method to prepare the high-performance membrane by constructing the directional proton channels with functionalized nanofibers. To solve the tradeoffs between ion selectivity and conductivity, amphoteric functionalized nanofibrous network along with an orientated ion channel was constructed, and the interface layer occupied with acid-base pairs generates good structural stability, chemical stability and excellent VRFB performance. • Amphoteric functionalized nanofibrous network (SP@f) contains 3D interlaced structure. • Nanofiber interface layer formed by acid-base pairs provides high concentration of proton carriers. • Ultra-high proton conduction depends upon the long and continuous proton transport channel. • S/SP@f-10 membrane shows outstanding battery efficiencies and long-term cycle stability.