Multilayered microstructures can accelerate the proton conduction process in proton exchange membranes (PEMs). Herein, we design and construct PEMs with microstructures based on bifunctional nanofibers and sulfonated poly(ether ether ketone) (SPEEK) nanofibers. Specifically, the bifunctional nanofibers composed of poly(vinyl alcohol) and chitosan are prepared and then combined with the electrospun SPEEK nanofibers. The stable microstructure is derived from the compatible interfacial property of nanofibers and the formed hydrogen bonds. The multilayered microstructure consisting of nanofibers accelerates the proton conduction even at subzero temperature because of regulating the proton conduction pathways. Specifically, the (SKNF/CPNF/SKNF)/PA membrane exhibits the proton conductivities of (0.951 ± 0.138) × 10-2 S/cm at -30 °C and (7.32 ± 0.37) × 10-2 S/cm at 160 °C. Additionally, the fine proton conductivity stability is demonstrated by the proton conductivity in the long-term test and the cooling/heating cycle test, such as 1.67 × 10-2 S/cm at -30 °C (after 1000 h), 4.52 × 10-2 S/cm at 30 °C (after 810 h), 1.12 × 10-2 S/cm at -30 °C, and 1.01 × 10-1 S/cm at 30 °C in the cooling/heating process (5 cycles). The single fuel cell possesses an open-circuit voltage of 0.886 V and a peak power density of 0.508 W/cm2 at 130 °C.