The widespread human exposure to perfluoroalkyl acids (PFAAs) has led to increasing public concern. In this study, we present a comprehensive measurement of total fluorine (TF), extractable organic fluorine (EOF), identified organic fluorine (IOF, total concentration of identified PFAAs quantified as fluorine) and 11 target PFAAs in human serum (n = 60), hair (n = 49) and nails (n = 39) collected from non-occupation exposed volunteers in 10 cities of Guangdong Province, China. The results indicated that EOF was the major form of fluorine in serum, accounting for 70–80% of TF. The levels of IOF contributed less than 10% of EOF. Perfluorooctane sulfonic acid (PFOS) was found to be the dominant PFAA with mean concentration of 23 ng·mL⁻¹ in serum, 35 ng·g⁻¹ in hair and 33 ng·g⁻¹ in nail, respectively. Short-chain PFAAs (C ≤ 10) were the predominant PFAAs in three matrices. Levels of PFOS, perfluorohexane sulfonic acid (PFHxS), perfluoroundecanoic acid (PFUdA) and perfluorotridecanoic acid (PFTrDA) in males are significantly higher than those in females (p < 0.01). Significant positive correlations were observed between nail and serum for PFOS (p < 0.01), perfluorooctanoic acid (PFOA) (p < 0.05) and PFHxS (p < 0.01), suggesting that human nails, a noninvasive sample, are a promising bio-indicator for PFAA risk assessment.