We report, for the first time, the application of fluorinated hexagonal boron nitride nanosheets (F-BNNs) as an effective inorganic matrix for matrix-assisted laser desorption and ionization mass spectrometry (MALDI-MS) analysis of perfluoroalkyl acids (PFAAs). Fluoride modification of F-BNNs increases both enrichment ability and ionization efficiency. The method was validated using environmental water, milk, human serum samples, and zebrafish imaging that has been previously exposed to PFAAs. The method provided in this work holds considerable promise in term of rapid analysis, sample requirement, and practicability.