Increasing production and use of per- and polyfluoroalkyl substances (PFASs) has been reported from China, and a few studies have shown there are subpopulations in China with high and increasing exposure to these chemicals. In this paper, we present a comprehensive exposure assessment of PFASs in fishery employees from Tangxun Lake, China. Exceptionally high serum concentrations of C4 to C12 PFASs were observed in fishery employees (n = 39, median perfluorooctanesulfonic acid (PFOS) 10 400 ng/mL) compared to a reference group from the same city (n = 9, median PFOS 18.7 ng/mL). On the basis of the comparison of different exposure pathways, it was concluded that contaminated fish from Tangxun Lake was the primary source of PFAS exposure to fishery employees, and there was a positive association between serum PFAS concentrations and time of employment in the fishery. PFOS isomer profiles in fishery employees showed a significantly higher proportion of linear PFOS (78.4%) compared to the background-exposed reference group (66.8%), reflecting the highly linear PFOS isomer profile (>90%) of lake fish. Median renal clearance rates (CLrenal) of C4 to C10 perfluoroalkyl carboxylic acids (PFCAs) and perfluoroalkanesulfonic acids (PFSAs) ranged from 0.020 to 16.5 mL/day/kg and 0.013 to 9.43 mL/day/kg, respectively. PFCAs with less than eight perfluoroalkyl carbons were primarily eliminated via urine, whereas other routes of excretion may have contributed to the elimination for long-chain PFCAs and PFSAs. Calculated daily PFOS exposures of fishery employees significantly exceeded tolerable daily intake limits, but clinical blood chemistry parameters were mostly within normal reference ranges. However, additional epidemiological studies are needed to address potential associations between PFAS exposure and health effects in the Tangxun Lake area.