In this study, a novel method for the analysis of perfluorinated compounds in whole blood has been developed and validated. The method was developed by using a conventional reversed-phase C18 column as a trapping column for the elimination of background contamination and high-performance liquid chromatography with tandem mass spectrometry for the target compounds analysis. The trapping column provided fast online separation of the background contamination of perfluorinated compounds. In this developed method, the limits of detection for different perfluorinated compounds ranged from 0.06 to 0.14 ng/mL. It is notable that the limit of detection (0.07 ng/mL) for perfluorooctanoic acid was improved significantly after the elimination of background contamination. The method was also validated in terms of sensitivity, accuracy, and precision. The recoveries ranged from 66.8 to 111.9%, with relative standard deviations from 2.1 to 15.3%. Our preliminary data suggest that the novel method based on trapping column cleanup followed by high-performance liquid chromatography with tandem mass spectrometry could be applied in studies on the human exposure to perfluorinated compounds.