Rationale Poly‐ and perfluoroalkyl substances (PFASs) and organophosphorus flame retardants (OPFRs) are two types of emerging organic pollutants with potential human health hazards. Here, a rapid and sensitive method was developed for the determination of sixteen PFASs and seven OPFRs in human serum by ultra‐performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS). Methods After optimizing the chromatographic and mass spectrometric conditions, respectively, 100 μL of serum sample was liquid–liquid extracted using 8 mL of methyl tert‐butyl ether. The 23 targets were quantified within 8 min. All targets were quantified by the isotope‐internal standard method in both negative‐ and positive‐ion mode by UPLC/MS/MS. Results The method was validated in terms of sensitivity, linearity, precision, and accuracy. The limit of detection ranged between 0.004 and 0.650 ng/mL. Recoveries ranged from70.0% to 118.9% with a relative standard deviation lower than 20%. The developed method was successfully applied to analyze targeted analytes in human serum samples. A total of 13 of 23 analytes were detected in over 50% of samples. Conclusions A sensitive and rapid method was developed to quantify sixteen PFASs and seven OPFRs in serum. Sensitivity, linearity, recovery, and precision were validated and found to be satisfactory. This method can be a valuable tool for evaluation of exposure to both PFASs and OPFRs with high separation efficiency and sensitivity.