The abuse of fentanyl and its analogues, which are potent, short-acting, synthetic narcotic analgesics, has become a major issue. Many cases containing fentanyl-analogue and novel synthetic opioids have also been reported. Hence, we report the determination of fentanyl, fentanyl-analogue and novel synthetic opioids in whole blood by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method is characterized by the use of a simple, fast and inexpensive liquid-liquid extraction (LLE) for sample preparation, a rapid run time (8 min) and a low required volume of whole blood (100 μl). The limits of detection (LODs) ranged from 0.005 to 0.03 ng/ml, and the lower limits of quantitation (LLOQs) ranged from 0.05 to 0.2 ng/ml. The method was shown to be liner over a concentration range of 0.2-40 ng/ml for fentanyl, norfentanyl and norcarfentanil and 0.05-40 ng/ml for all other target analytes. Recoveries were within the range of 72.09%-103.22%, and the matrix effects were in the 67.95%-113.32% range. Moreover, the method was applied to the detection and quantification of fentanyl and its analogues in whole blood from real forensic cases. This methodology has great potential for use in the determination of fentanyl and its analogues in forensic cases.