Abstract: 100 μL of 100 μg·L^-1 fentanyl-d₅ internal standard solution and 4 mL of 0.1 mol·L^-1 hydrochloric acid solution were added into 1.0 mL of urine or blood sample. The mixture was oscillated and centrifuged, the supernatant was passed through activated MCX solid phase extraction column, and the eluent was blown to near dryness by nitrogen. The residue was dissolved in a mixture of 5 mmol·L^-1 ammonium acetate buffer solution including 0.1% (φ) formic acid and acetonitrile at volume ratio of 9:1, and its volume was made up to 1 mL. The solution was filtered through a 0.22 μm organic filter membrane, and 15 fentanyl-analogue new psychoactive substances were determined by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) with ACQUITY UPLC BEH C₁₈ column as the stationary phase and the mixtures of 5 mmol·L^-1 ammonium acetate buffer solution including 0.1%(φ) formic acid and acetonitrile at different volume ratios as the mobile phase for gradient elution. Electrospray ion source positive ion (ESI⁺) mode and multiple reaction monitoring (MRM) mode were adopted in MS analysis. As shown by the results, linear ranges of standard curves of 15 fentanyl-analogue new psychoactive substances were 0.5-50.0 μg·L^-1, with the same detection limits (3S/N) of 0.1 μg·L^-1. Tests for the spiked recovery and inter-day (n=6) and intra-day (n=6) precision were made on blank samples, giving results in the range of 81.1%-113%, with RSDs of the determined values less than 15%.