Abstract: Objective To develop a method for the determination of nicotine and cotinine in urine samples by dispersive liquid-liquid microextraction (DLLME) combined with gas chromatography-mass spectrometry (DLLME-GC/MS). Methods The experimental conditions for GC-MS and DLLME were investigated in detail. DLLME was performed with the following procedure: 5 mL of urine sample was adjusted to pH9.0 with NaOH solution; NaCl was added to increase ionic strength; 100 μL chloroform (containing internal standard of quinolone) as extractant was mixed with 1 000 μL methanol as dispersant and then injected into the urine sample to make it emulsified and dispersed. The sample solution was centrifuged for 5 min at 4 000 r/min, and 1 μL of its extraction solvent was injected into the GC/MS system for analysis. GC separation was performed with DB-5 column under programmed temperature. Nicotine and cotinine were quantified using selected ion monitoring (SIM) mode of mass spectrum detection and internal standard working curve. Results Good linear relationship was obtained for detecting nicotine and cotinine ranging from 0.2 ng/mL to 100 ng/mL, with a detection limit of 0.010 ng/mL and 0.022 ng/mL for nicotine and cotinine respectively. The relative standard derivation (RSD) for determination of nicotine and cotinine in urine samples were 8.2% and 9.6% respectively. The spiked recoveries ranged from 92.0% to 108.0% for nicotine, 83.0% to 110.0% for cotinine. Conclusion The method is rapid, sensitive, accurate and simple, with little consumption of organic solvent. It is suitable for determination of nicotine and cotinine in urine, and can meet the requirements for evaluating human tobacco exposure.