Assessment of the exposure risk of perfluoroalkyl and polyfluoroalkyl substances (PFASs) has been of public concern for many years. However, it is a challenging undertaking because of the trace levels of these contaminants in the environment and biological systems. In this work, fluorinated carbon nanotubes/silk fibroin (F-CNTs/SF) nanofibers were for the first time synthesized by electrospinning and evaluated as a new adsorbent in pipette tip-solid-phase extraction to enrich PFASs. The addition of F-CNTs increased the mechanical strength and toughness of the SF nanofibers, thus improving the durability of composite nanofibers. The proteophilicity of silk fibroin formed the basis of the good affinity of this material with PFASs. The adsorption behaviors of PFASs on the F-CNTs/SF were investigated by adsorption isotherm experiments to understand the mechanism of extraction. With analysis using ultrahigh performance liquid chromatography-Orbitrap high-resolution mass spectrometric, low limits of detection (0.006–0.090 μg L−1) and enrichment factors of 13–48 were obtained. Meanwhile, the developed method was successfully applied to the detection of wastewater and human placenta samples. This work provides a new idea for the design of novel adsorbents with proteins integrated in polymer nanostructures, a potential routine and practical monitoring technique for PFASs in environmental and biological samples.