A method was developed for high throughput and high sensitive detection of free triiodothyronine（FT3） and free thyroxine（FT4） in serum using gel filtration chromatography（GFC） and liquid chromatography-tandem mass spectrometry（LC-MS/MS）. The hydroxypropyl cellulose gel Sephadex LH-20 column was used to filter and separate 150 μL serum samples. First of all，the thyroid hormones bound to binding proteins were removed by washing with tri（hydroxymethyl） aminomethane hydrochloride（Tris-HCl） buffer solution（0.1 mol/L，pH 7.4）. Following that，the free thyroid hormones were eluted using methanol. The established LC-MS/MS analysis method was employed for detection and quantification. The results of the methodological validation showed that the correlation coefficient（,r,2,） was not less than 0.999 5. The precision ranges from 1.8% to 10%，accuracy ranges from 85.4% to 110%，matrix effect ranges from 85.8% to 114%，and recoveries from 85.8% to 107%. According to the CLSI EP09-A3 method comparison guidelines，this method was further applied to the detection of 95 serum samples and compared with the equilibrium dialysis（ED），to evaluate the accuracy and applicability of the established GFC/LC-MS/MS method. The statistical analysis was performed using IBM SPSS Statistics 26 and MedCalc statistical software（v.20.0.0） for paired ,t,-test analysis，Passing-Bablok regression analysis，and Bland-Altman agreement analysis. The statistical analysis results indicated that there was no significant difference（,P,>,0.05） between the detection results of FT3 and FT4 in the serum measured by the developed GFC/LC-MS/MS method and the ED/LC-MS/MS method. The developed method can provide a reference method for rapid，efficient，and accurate detection of free thyroid hormones in clinical laboratories，as well as offering a new approach for the detection of other free hormones such as cortisol and testosterone.