A supercritical fluid chromatography （SFC） method coupled with UV detection was developed for the separation of linagliptin and its S-enantiomer. The method was validated and successfully applied to detect the S-enantiomer in real pharmaceutical samples. The separation of the enantiomer was investigated using six different chromatographic columns， and different cosolvents were studied. Chromatographic conditions， including column temperature， backpressure， and flow rate， were optimized. The DAICEL CHIRALPAK AD-H column （250 mm×4.6 mm， 5 μm） was used for separation. Supercritical CO₂ served as mobile phase A， and ethanol-isopropanol （1∶1， volume ratio） containing 0.25% diethanolamine and 0.25% trifluoroacetic acid was used as mobile phase B. Isocratic elution was carried out at a ratio of A∶B=73∶27 （volume ratio） with a flow rate of 1.5 mL/min. The column temperature was set at 40 ℃， back pressure at 15 MPa， injection volume at 6 μL， and detection wavelength at 220 nm. Under these conditions， linagliptin and its S-enantiomer were separated with a resolution of 3.1 and good peak shapes. Both linagliptin and its S-enantiomer exhibited good linearity in the concentration range of 2–90 μg/mL， with correlation coefficients of 0.999　7 and 0.999　9 （n=8）， respectively. The limit of detection （LOD） for both was 0.8 μg/mL （S/N=3）， and the limit of quantification （LOQ） for both was 2 μg/mL （S/N=10）. The average recoveries of the S-enantiomer spiked at low， medium， and high concentrations in active pharmaceutical ingredients and tablets were 97.4% （RSD=1.1%， n=9） and 101.6% （RSD=1.2%， n=9）， respectively. S-Enantiomer was not detected in three batches of active pharmaceutical ingredients or in three batches of tablets from two different manufacturers. This study represents the first application of SFC for the separation of linagliptin and its S-enantiomer. The method is environmentally friendly， sensitive， and highly efficient， offering good repeatability of peak area. It provides a solid foundation for the quality control of linagliptin and the inclusion of the SFC method in pharmaceutical quality standards， while also offering a useful approach for the rapid separation and impurity control of other chiral drugs.