Abstract: To determine the 3 steroid estrogens, including estrone, estradiol, and estriol in serum, while simplifying the pretreatment process, reducing solvent consumption and human operation errors, the method mentioned by title was proposed. 200 μL of serum sample was taken and placed in a 1.5 mL-centrifuge tube. Then, 600 μL of 2 μg·L⁻¹ mixed internal standard working solution was added, and the mixture was vortexed for 60 s and centrifuged for 10 min. 200 μL of supernatant was collected and introduced into 800 μL of water, and the mixture was vortexed for 60 s. The resulting solution was introduced into the online solid phase extraction system, and alternately loaded and purified on two Oasis HLB Direct Connect HP solid phase extraction columns, then the extract was introduced into the ultra-high performance liquid chromatography-tandem mass spectrometry system. 3 targets were separated on Kinetex Polar C₁₈ column using a gradient elution system consisting of 0.5 mmol·L⁻¹ ammonium fluoride solution and acetonitrile. Quantification was performed in modes of negative electrospray ionization and multiple reaction monitoring using the internal standard method. As shown by the results, linear relationships between the ratios of the corresponding peak area to the internal standard peak area and mass concentrations of 3 targets were kept in the range of 0.01−50 μg·L⁻¹, with detection limits （3S/N） in the range of 0.50−0.73 ng·L⁻¹. Test for recovery was made by the standard addition method, giving results in the range of 97.6%−106%, with RSDs （n=6） of the determined values in the range of 1.2%−6.6%. This method was applied to the analysis of 30 serum samples, and estrone, estradiol and estriol were all detected, with detection amounts in the range of 15.31‒95.78 ng·L⁻¹, 2.14‒123.32 ng·L⁻¹ and 3.06‒29.08 ng·L⁻¹, respectively.