Abstract: Objective To develop a QuEChERS-gas chromatography method for simultaneous determination of nicotine and cotinine in urine samples.Methods Urine samples were collected from 6 smokers and 10 non-smokers at Chongqing Medical University;then the samples were extracted and purified with quick,easy,cheap,effective,rugged,and safe (QuEChERS) method and then separated via a non-polar DB-5 ms capillary chromatographic column and determined with flame ionization detector (FID).Qualitative and quantitative analyses were performed using retention time and peak area.Results The linear ranges of nicotine and cotinine for the detected samples were 0.5-500 μg/mL (r=0.9998) and 5-500 μg/mL (r=0.998 9),with the limit of detection (LOD) of 0.09 μg/mL and 0.75 μg/mL,respectively.The intra-day and inter-day relative error (RE) and coefficient of variation (CV) of nicotine and cotinine detections were less than 10.0% and 10.4%.The mean recoveries of nicotine and cotinine were 93.2%-107.8% and 94.6%-99.2%,respectively.Conclusion The method developed is simple,fast,cost-effective,and suitable for the assessment of tobacco exposure dose and the detection of metabolic dynamics of tobacco exposure.