Organophosphate esters (OPEs) are widely used as flame retardants and plasticizers, which are of growing concern due to their endocrine-disrupting effects, developmental toxicity, and potential carcinogenicity. However, data on human exposure to OPEs is still scarce. In this study, a relatively simple and efficient method with less serum consumption for the detection of OPEs in human serum was developed and validated. Nine OPEs in 200μL of human serum were extracted by an acetonitrile–formic acid system and analyzed using ultra-high-performance liquid chromatography–quadrupole tandem time-of-flight high-resolution mass spectrometry. Several experiments were conducted to optimize the chromatographic and mass spectrometric conditions as well as sample preparation to obtain a more sensitive and efficient analytical protocol. The proposed method was examined in terms of its linearity, accuracy, precision, detection limit, and matrix effect. The matrix-spiked recoveries of the target OPEs ranged from 83.3 % to 111.1 %, with relative standard deviations between 2.7 % and 16.6 %. The detection limits were within (0.002 to 0.029) ng/mL, while the quantification limits were within (0.007 to 0.098) ng/mL. The internal standard-corrected matrix effects varied from 82.7 % to 113.9 %. Finally, the method was applied to detect OPEs in actual human serum samples. All nine OPEs were detected in 269 serum samples to varying degrees, with the average concentrations ranging from (0.08 to 1.77) ng/mL. After validation, the method was found to be simple in pretreatment, high in sensitivity, good in practicality, and suitable for exposure evaluation of OPEs in populations.