The safety of lithium-ion battery is closely related to the anti-shrink and heatproof stability of the separator. In this paper, electrospun nylon66 (PA66) nanofiber-based membrane is used as lithium-ion battery separator, showing good thermodynamics properties via thermogravimetric analysis (TG), thermal shrinkage experiment, and tension test. Furthermore, electrospun nylon66 separator based battery exhibits better safety than the battery applying Celgard commerce separator under the condition of high temperature and severe vibration. Meanwhile, the electrochemical properties of both batteries are nearly identical. These facts prove that the electrospun nylon66 separator is an ideal separator candidate for power lithium-ion battery of electric vehicles. In addition, the nylon66 separator annealed in the air has higher tensile strength and better property of elongation than the unannealed one.