Abstract: A method was established for the determination of fentanyl and its metabolite (norfentanyl) in urine by on-line SPE combined with UHPLC-MS/MS, which was applied to the study of detection window of fentanyl and its metabolite in rat urine. The urine was diluted with 2 mmol·L^-1 ammonium formate buffer solution at pH 9.2, and the diluted sample was directly injected after centrifugation. The target compounds were enriched and purified by on-line SPE with Oasis HLB solid phase extraction column, and eluted onto the Hypesil GOLD C₁₈ analytical column with the initial mobile phase of the analytical pump. A mixture of methanol solution containing 2 mmol·L^-1 ammonium formate and 0.1% (φ) formic acid, and aqueous solution containing 2 mmol·L^-1 ammonium formate and 0.1% (φ) formic acid in different ratios was used as the mobile phase for gradient elution. ESI⁺ and the mode of selected ion monitoring (SRM) were adopted in MS/MS. Rat urine samples were collected after the tail vein injection fentanyl of rats at a dose of 20 μg·kg^-1, and the detection window of the protosome and metabolite was studied by this method. As shown by the results, linear relationships between values of peak area and mass concentration of fentanyl and norfentanyl were found in the same range of 2.00-800 ng·L^-1, with the same detection limit (3S/N) of 0.200 ng·L^-1. RSDs (n=5) of the determined values were all less than 15%. The detection window of protosome fentanyl in rat urine was 5-7 d, while the detection window of its metabolite norfentanyl could be up to 28 d.