This paper presents for the first time that poly( l ‐lactic acid) (PLLA) nanofibers can show the piezoelectricity along the fiber direction (d 33 ) by using an electrospinning method. First, the electrospun fiber bundles are characterized by scanning electron microscope, X‐ray, and piezoelectric coefficient measurements. The data show that the supercritical CO 2 treatment can greatly enhance the piezoelectricity of electrospun PLLA fibers, which can be resulting from the increased crystallinity of the fibers. Later, it is found that the electrospun PLLA fiber can generate a current of 8 pA and a voltage of 20 mV by a simple push–release process. Further, a single PLLA fiber‐based blood pulse sensor is also fabricated and tested and shows around a 2 pA output for blood pulse. Due to easy fabrication and relatively simple structure, this device enables a broad range of promising future applications in the medical sensor area. image