Abstract: A kit and a method for determination of fentanyl drugs in biological samples are provided, belonging to the field of biotechnology. The method includes: a sample is transferred into a centrifuge tube containing acetonitrile in advance for shaking, extraction and centrifugation; a supernatant is drawn into a purification extraction column by pulling a plunger upwards and fully contacted with absorbents to quickly complete a preliminary purification; the plunger is pulled upwards continuously to absorb a certain volume of air, then a filter is installed at the bottom of the purification extraction column and the plunger is pushed downwards to make a sample extractant comes into contact with mixed absorbents. The filtrate is collected and subjected to analysis on liquid chromatography-tandem mass spectrometry.