Abstract: Traditional and emerging per- and polyfluoroalkyl substances (PFASs) are persistent organic pollutants that have attracted increasing attention due to their potential health risks to humans. Therefore, the method of simultaneous determination of 7 perfluorocarbonic acid compounds, 3 perfluorosulfonic acid compounds, and 2 novel PFASs substitutes perfluoro (2-methyl-3-xahexanoic) acid (HFPO-DA), 6∶2 chlorinated polyfluoroalykl ether sulfonate (6∶2 Cl-PFESA) in serum by ultra-high performance liquid chromatography-tandem mass spectrometry was proposed. After thawing, healthy human serum samples were taken, and mixed internal standard solution and the acetonitrile-methanol mixted solution at volume ratio of 1∶1 were added. After mixing by ultrasound, the mixed solution was frozen at －20 ℃ for 1 h, and centrifuged at high speed and 4 ℃, and the supernatant was freeze-dried at the vacuum condition and dissolved in the methanol-water mixed solution at volume ratio of 3∶7. The obtained solution was mixed by vortex and sonicated in an ice bath. After centrifugation at high speed and 4 ℃, the supernatant was determined by the intrument. The 12 PFASs were separated by gradient elution with the mixed solution of 2 mmol·L⁻¹ ammonium acetate solution and methanol at different volume ratios on Phenomenex Kinetex F5 column, ionized by negative ion mode of electrospray ion source, detected by multiple reaction monitoring mode, and quantified by internal standard method using fetal bovine serum as matrix. It was shown that linear relationships between values of the mass concentration ratio and the peak area ratio of the target to internal standard were kept in definite ranges, with detection limits (3S/N) in the range of 0.005－0.01 μg·L⁻¹. Test for recovery was made at 3 concentration levels, giving recoveries ranging from 88.5% to 112%, and RSDs (n＝5) of the determined values were found in the range of 0.20%－8.7%. The proposed method was used for the analysis of 20 serum samples from healthy individuals, with detection rates of 100% for 9 PFASs (including a novel PFASs substitute of 6∶2 Cl-PFESA).