Abstract: Per- and polyfluoroalkyl substances (PFAS) represent a class of persistent, bioaccumulative, and potentially toxic emerging environmental contaminants, making their precise quantification in biological organisms essential for health risk assessments and policy-making. This paper focuses on sample preparation techniques for the chromatographic analysis of PFAS in biological samples, systematically reviewing the latest research progress and current challenges. Addressing core analytical hurdles—including complex matrices, trace concentrations, significant matrix effects, and background contamination—the study elucidates the critical role of tailored sample preparation in achieving analyte purification, enrichment, and improved analytical efficiency and accuracy. Finally, the paper envisions a future centered on automated, miniaturized, and "green" pretreatment methods integrated with high-resolution mass spectrometry, providing a scientific foundation for PFAS source tracking, toxicological research, and risk management.