Abstract: New psychoactive substances of fentanyl are opioid pain reliever with strong hallucinogenic, excitatory, and narcotic effects. Fentanyl and its derivatives became alternatives to traditional drugs and were exploited by criminals for profit. Rapid detection of fentanyl drugs has become the primary task of forensic expertise. Direct analysis in real time-tandem mass spectrometry (DART-MS/MS) was used to establish a rapid screening method on 8 kinds of fentanyl in human whole blood and urine. Firstly, 100 μg/L fentanyl standard was used for mass spectrometry optimization to determine the mass spectrum conditions of 8 kinds of fentanyl. Then the temperature of DART ion source was optimized, and the temperature of DART ion source was determined to be 450 ℃. Methanol-acetonitrile solution (1∶4, V/V) was used for the pretreatment of blood and urine samples. Then the samples were centrifuged and supernatant was extracted for sampling by the DART 12Dip-it automatic sampling system. The results showed that the linear relationship was good in the concentration range of 50-800 μg/L, the correlation coefficients ranged from 0.992 1 to 0.999 6, the limits of detection were all less than 0.05 μg/L, the relative standard deviations (RSDs) of repeatability were all less than 10%, and the RSDs of intra-day and inter-day precision were all less than 15%. The method has the characteristics of high sensitivity, convenience and small pollution, which can provide data support for the rapid detection of new psychoactive substances associated with fentanyl in biometric materials in judicial practice.