Abstract: OBJECTIVE To explore and establish an analysis method for sulfamethoxazole blood concentration in patients with heart transplantation to guide clinical rational drug use. METHODS A new two-dimensional liquid chromatograph (2D-LC-UV) based on column switching technology was used. The on-line solid-phase extraction of sulfamethoxazole in serum was performed using a one-dimensional column Aston SC2(3.5 mm×25 mm, 5 μm), then it was intercepted and retained through the middle column Aston SBR(3.5 mm×10 mm, 5 μm), and the target analytes were completely separated and detected by transfer to the second-dimension column Aston SNX4(4.6 mm×130 mm, 5 μm). The chromatographic conditions were obtained by optimization. The one-dimensional mobile phase was acetonitrile-methanol-H₂O(10:10:70), the flow rate was 0.8 mL·min^-1; the two-dimensional mobile phase ratio was BPI-1 basic mobile phase-API-3 acidic mobile phase-methanol(20:40:40) the flow rate was 1.2 mL·min^-1. The UV detection wavelength was 240 nm. RESULTS The correlation coefficient between the concentration of sulfamethoxazole and peak area was in the range of 9.96‒200.04 μg·mL^-1 with R²=0.999 6, it showed a good linear relationship. The intra-day and inter-day precisions(RSD) at low, medium and high concentrations were <15%, and the relative recoveries were between 85%‒115%. The blood concentration of 56 patients in the hospital was measured, and only 30 patients(53.57%) had the blood sample sulfamethoxazole concentration of 100‒150 μg·mL^-1, and the rest were not comfortable about the concentration treatment window; there were 0 cases of grade III-IV adverse reactions in the urinary system/blood system/liver, etc. CONCLUSION This method has a simple pre-treatment, high automation, can be sampled in large volumes, and has high accuracy and sensitivity. It can meet the requirements of clinical applications. The research results can provide a methodological reference for clinical therapeutic drug monitoring..