In recent years, the occurrence of novel per-/polyfluoroalkyl substances (PFASs) such as polyfluoroalkyl ether sulfonates (PFAESs) in human samples have aroused attention due to the change in PFASs production profile, however, the data are still lacking. Furthermore, epidemiological studies have examined the associations of PFAS exposure with glucose homeostasis, but with inconsistent results. Therefore, in this study, fasting serum samples from 252 participants with an age range from 19 to 87 years old were collected in Tianjin, China. A total of 21 target PFASs were determined to analyze the levels and distribution of novel and legacy PFASs in serum and to further evaluate the cross-sectional associations of serum PFAS concentrations with two glycemic biomarkers (i.e., fasting glucose and glycated hemoglobin (HbA1c)). 6:2 chlorinated PFAES (6:2 Cl-PFAES) and trifluoroacetic acid (TFA) were widely detected novel PFASs (greater than90%) with relatively high median concentrations (8.64 ng/mL and 8.46 ng/mL, respectively), which were second only to the two dominant legacy PFASs, i.e., perfluorooctanoic acid (PFOA, 14.83 ng/mL) and perfluorooctane sulfonic acid (14.24 ng/mL). The percentage contributions to the total known PFASs were separately 17.6% and 17.2% for 6:2 Cl-PFAES and TFA. The levels of 6:2 Cl-PFAES were significantly correlated with age and BMI, and the concentrations of TFA were also significantly correlated with age. Furthermore, 1% increase in serum PFOA and perfluorononanoic acid (PFNA) was separately significantly associated with 0.018% [95% confidence interval (CI): 0.004%, 0.033%] and 0.022% (95% CI: 0.007%, 0.037%) increment in fasting glucose levels. Similarly, 1% increase in serum perfluorohexanoic acid, PFNA, and perfluorohexane sulfonic acid was significantly associated with 0.030% (95% CI: 0.010%, 0.051%), 0.018% (95% CI: 0.003%, 0.033%), 0.007% (95% CI: 0.003%, 0.011%) increment in HbA1c levels, respectively. These findings suggested that 6:2 Cl-PFAES and TFA showed greater contributions to PFASs in serum and supported an association of exposure to PFASs with fasting glucose and HbA1c.