Stimuli-responsive electrospun nanofibers of less than 1000 nm in diameter are a kind of smart polymer fibers prepared by electrospinning process, which can respond to external stimuli with the change of their physicochemical properties. The nanofibrous membranes consisting of the stimuli-responsive electrospun nanofibers have the advantages of large specific surface area, high porosity and fast stimuli-responsiveness, have attracted worldwide attention in recent years due to their fascinating application prospect within many fields. In this paper, three methods for preparing stimuli-responsive electrospun nanofibers are firstly summarized. Then, the main factors influencing the size, structure and stimuli-responsive property of the nanofibers are discussed from synthesis or selection of the fiber-forming polymers, preparation of spinning solution, electrospinning and post-treatment. Subsequently, the research progress on the designs and fabrications of all kinds of stimuli responsive electrospun nanofibers except electric field responsive nanofiber is reviewed, and their applications in separation and purification, drug controlled release, wound dressing, cell culture, sensor and detection are introduced. Finally, their future research direction is briefly commented.