Abstract: Silk fibroin (SF) solution is prepared using a formic acid/calcium chloride system, and SF fibers are fabricated through electrospinning technology, combined with post-stretching in an ethanol aqueous solution. The SF fiber is used as the piezoelectric layer, with aluminum electrodes as the positive and negative terminals, and polyimide (PI) tape is used for encapsulation, resulting in the development of a high-performance SF fiber-based piezoelectric nanogenerator (SF@PENG). The piezoelectric performance of SF@PENG is investigated using an oscilloscope and an electrostatic voltmeter, allowing for a comparative analysis of performance differences before and after the post-treatment. Furthermore, Fourier-transform infrared spectroscopy (FT-IR) and wide-angle X-ray diffraction (XRD) are utilized to analyze the structural changes of SF after the post-treatment. The results demonstrate that the post-treated SF@PENG achieves a maximum output voltage of 20.8 V and a maximum output current of 8.2 μA under an external force of 25 N, and exhibits stable cycling performance for over 9500 cycles under a constant external force of 21 N. Additionally, increasing the content of the β-sheet structure and crystallinity of SF significantly enhances the piezoelectric output properties of SF@PENG. SF@PENG is capable of monitoring different movement states of the human body, laying a solid foundation for applications in self-powered devices.