Understanding the microstructure of electrospun fibers is critical for tailoring fiber properties.The single-step acquisition of electrospun polyvinylidene fluoride fibers enriched with β-phase content is a promising energy material to replace butteries in small-scale flexible electronics. In this work, we focused on the evolution of the microstructure of electrospun PVDF fibers during stretching at varying temperatures and investigated the mechanism of electrospun fibers stretching in the view of the combination of fiber microstructure and macroscopic morphology considerations. The responses of alignment and structure, diameter, crystal orientation and crystal structure of electrospun fibers on the synchronous stimuli of temperatures and stretching were studied in detail. We found that at 150 °C, under the stimulation of stress, the fibers with lower alignment present higher molecular orientation, β-phase content reaching up to 90.5%, and excellent mechanical properties, which can be the main candidates for energy harvesting. Furthermore, not only macroscopic morphology but also the fiber microstructure is crucial for improving mechanical property of electrospun PVDF fibers membrane. The microstructure responses of electrospun PVDF fibers on such synchronous stimuli are positive for regulating the final properties of products, which also can open a new avenue for the design and exploitation of other electrospun fibers.