Per- and polyfluoroalkyl substances (PFASs) represent a group of synthetic chemicals, and they have quite different physicochemical properties, which result in difficulties of their simultaneous determination in a single injection. A sensitive, reliable, and fully automated method was developed for simultaneously detecting 10 classes of PFASs (total of 43) in human serum using online Turboflow SPE-UHPLC-MS/MS. This method provided high linearity of matrix-matched calibration standards (R > 0.99), excellent method limits of detection (MLODs) (0.013–0.089 ng mL −1 ), satisfactory matrix spiked recoveries (84.3–109%) and relative standard deviations (RSDs) (intra-day RSDs: 1.3–12.6%, inter-day RSDs: 1.7–13.8%, inter-week RSDs: 1.8–13.5%, inter-month RSDs: 3.1–12.4%), short analysis time (19 min per sample) and small sample amount requirement (25 μL), which were suitable for large-scale epidemiologic studies. Moreover, the method provided the feasibility of real-time monitoring for the degradation kinetics of PFASs precursors both in vitro and in vivo. The quality of the present method was further verified by repetitive analysis of a standard reference material (SRM 1957), with the deviations of the targeted PFAS concentrations ranging from 1.9% to 14.2% (n = 5) between the detected and reference values. The present study also determined values for several PFASs in SRM 1957 other than those on the certificate, for the first time, such as N-EtFOSA, 6:2 Cl-PFESA, and PFBA. Finally, the established method was applied to detect PFASs in serum samples of 15 ordinary people and 15 occupational workers, and 6:2 FTSA was found as the dominant precursor. • An automated online SPE method was developed for detecting 10 classes of PFASs in 25 uL serum. • PFASs detected by the present method were consistent with the reference values of SRM 1957. • Several PFASs in SRM 1957 without reference concentrations were determined for the first time. • The method is promising for epidemiologic studies and degradation kinetics studies of PFASs precursors.