The invention relates to a liquid chromatogram-tandem mass spectrum inspection method for oleandrine and oleandrine B in biological fluid. The liquid chromatogram-tandem mass spectrum inspection method comprises the following steps: cutting up a liquid sample or a tissue sample and putting in a test tube with a stopper; taking the prepared sample, adding ethyl acetate twice, and oscillating for 10 minutes and centrifuging for 10 minutes each time; mixing organic phases obtained after oscillating and centrifuging twice, concentrating into 0.5mL by virtue of an air concentrator, and filtering through an organic filtration membrane with 0.22micron micropores; and carrying out liquid chromatogram-tandem mass spectrum analysis on filtrate. The detection method provided by the invention is simple, efficient, fast, sensitive, high in accuracy and wide in practicability, can be applied to quantitative and qualitative inspection of the oleandrine and the oleandrine B in a biological sample, and is suitable for inspection of in vitro samples and suspicious physical evidences.