Abstract: An analytical method with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was established to test uniconazole residues in Gossypium spp. leaves, Gossypium spp. seeds and soil. Uniconazole residue and dissipation on Gossypium spp. leaves and soil were also investigated with this method. Uniconazole residues were extracted with acetonitrile/water mixture and an aliquot of the extract was purified by a solid-phase extraction Cleanert NH2 cartridge. Finally, the analytes were measured by multiple reaction monitoring (MRM) in the electrospray positive ionization mode. The external standard method was adopted for the quantification. The results showed that the recovery rate of uniconazole in Gossypium spp. leaves, Gossypium spp. seeds and soil were between 74%-101%, with the relative standard deviations from 1.2% to 10% (n = 5). The limits of detection (LOD) of the method were 0.01 ng and the limits of quantification (LOQ) were 0.01 mg/kg for three matrices. The method is accurate, sensitive, simple and suitable for the determination of uniconazole residues in Gossypium spp. samples. Field experimental results showed that the dissipation half-lives of uniconazole in Gossypium spp. leaves and soil were 4.2-5.0 days and 15.8-19.7 days, respectively. The terminal residues at harvest in Gossypium spp. seeds and soil were lower than 0.01 mg/kg and 0.022 mg/kg, respectively.