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[en] In this study, we prepared a novel type of biofunctionalized poly(vinylidene fluoride) (PVDF) nanofibers. Cysteine, a natural amino acid, was grafted onto the surfaces of PVDF nanofibers, The −SH groups of cysteine were then oxidized to −SO3H. The formation of proton-conducting pathways was induced by the −SO3H and COOH groups of the oxidized cysteine chains on the surfaces of the biofunctionalized PVDF nanofibers. Composite membranes were fabricated by impregnating the biofunctionalized PVDF nanofibers with Nafion. Then, the effects of incorporating nanofibers grafted with different amounts of oxidized cysteine on the thermal stability, water uptake dimensional stability, proton conductivity, and single-cell performance of Nafion composite membranes were investigated. The properties of the composite membranes were superior to those of the Nafion membrane. Furthermore, Nafion/PVDF–Cys-30 exhibited the highest proton conductivity of 0.22 S cm−1 (80 °C), and the maximum power density of 108.42 mW cm−2 which was twice than the values of Nafion 117 membrane (51.2 mW cm−2) at 60 °C under 100% RH. The introduction of biofunctionalized nanofibers significantly improved cell performance, proton conductivity, dimensional stability, and methanol permeability of the membrane.