Fluorous solid-phase extraction (FSPE) has been employed to isolate target compounds from complex chemical or biological samples in many research areas. However, the lack of efficient and economical available perfluorinated materials impeded its development. In this study, we present a novel nanographite fluoride-based fluorous solid-phase extraction (GF-FSPE) as a replacement of commercially available cartridge-based FSPE, which showed remarkable selectivity, low LOD, high post enrichment recovery, and large enrichment capacity. To demonstrate the potency of GF-FSPE, it was designed and successfully applied to isolate cysteine-containing peptide subsets from complex protein samples to improve the proteome coverage. Additionally, since graphite fluoride was inexpensive and highly commercialized, this study was expected to promote the popularization of FSPE in both chemical and biological separations as well as encourage the synthesis and broaden the application of highly fluorinated carbon fluoride in material science.