Abstract: In order to quantify the nicotine and cotinine levels in human urine, an automatic online thermal desorption-gas chromatography/mass spectrometry (GC/MS) method was developed. By using steam-treated SBA-15 type mesoporous silica as the sorbent and coupling with GC/MS, a device was designed for the efficient enrichment and rapid online analysis of nicotine and its metabolite cotinine in urine. The results showed that the device realized the online sample adsorption, thermal desorption, injection and analysis. The SBA-15 type mesoporous silica modified with steam at 700℃ for 6 h significantly improved the thermal desorption of nicotine and cotinine. Calibration curves in the range of 1-2 000 ng/mL of the analytes were established with R² higher than 0.999 5. The spiked recoveries of the analytes ranged from 97.28% to 104.55% with the relative standard deviations (RSDs) of 0.25%-3.18%. The limits of detections for nicotine and cotinine were 0.5 and 0.4 ng/mL, respectively. This method is highly automatic, rapid and suitable for the analysis of nicotine and cotinine in human urine samples.