Polytetrafluoroethylene (PTFE) nanofiber membranes with a kind of novel circular pore structure were fabricated in our study. Firstly, PTFE/PVA spinning solution was electrospun into precursor nanofiber membrane, during which, poly(tetrafluoroethylene-hexafluoropropylene) (FEP) aqueous dispersion was introduced by electrostatic spraying aiming to optimize the original fibrous network pore structure of electrospinning. After which, the prepared precursor nanofiber membranes were sintered, and PTFE nanofiber membranes were finally obtained. Effects of FEP contents on PTFE nanofiber membranes' structure and performance were investigated, respectively. From the morphology observation, it was found that the PTFE nanofiber membrane's pore structure gradually transformed from fibrous network to circular pore due to the melting of FEP resins. Owing to the excellent under-oil hydrophobicity and the uniform circular pore structure, the obtained PTFE nanofiber membranes were utilized in membrane emulsification (ME) process to prepared water in oil (W/O) emulsion. Compared with the conventional mechanical stirring method, the W/O emulsion prepared by ME process showed not only more uniform water droplet size (about 270 nm), but also more stable and higher production efficiency.