Objective To compare the results of high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) and chemiluminescence immunoassay (CLIA) in the determination of serum estradiol (E2) level. Methods From January 2023 to January 2024, 100 patients who were treated in outpatient department and hospitalized in the hospital were selected as the research subjects. Fresh blood samples were collected from all study subjects on the same day, and serum E2 levels were measured by LC-MS/MS and CLIA respectively. Results Serum E2 level measured by CLIA was significantly higher than that of LC-MS/MS ($P <{0.05}$). Pearson correlation test results showed that LC-MS/MS was significantly correlated with CLIA in measuring serum E2 level$\left({r ={0.866}, P <{0.05}}\right)$. There was a linear correlation between serum E2 level measured by LC-MS/MS and CLIA, and the linear regression equation was manifested as$Y ={1.161X}-{6.574}$ $\left({r ={0.973}, n={100}}\right)$. Bland-Altman analysis revealed that the average difference between LC-MS/MS and CLIA in measuring serum E2 level was${0.078}\mathrm{{nmol}}/\mathrm{L}\left({{95}\%\mathrm{{CI}}:{0.069}\sim {0.086}\mathrm{{nmol}}/\mathrm{L}}\right)$, and the consistency limit range was (-0.011,0.143). There were 5%(5/100) points outside the 95% consistency limit range, and within the 95% consistency limit range, and the maximum difference between LC-MS/MS and CLIA was${0.156}\mathrm{{nmol}}/\mathrm{L}$. The average value of serum E2 level measured by LC-MS/MS and CLIA was${0.152}\mathrm{{nmol}}/\mathrm{L}$, suggesting that the consistency of the two methods was poor. Conclusion Serum E2 level determined by CLIA is higher than that determined by LC-MS/MS. There is a certain correlation between the two methods for the determination of serum E2 level, but the consistency is not good. The determination of serum E2 level by CLIA is more suitable for rapid screening. When accurate clinical diagnosis is needed, it is necessary to apply LC-MS/MS to measure serum E2 level.