An analytical method for the determination of glyphosate (GLY) and aminomethylphosphoric acid (AMPA) in biological fluid samples (serum and urine) from poisoning patients using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is established. After the sample pretreatment, including protein precipitation and a modified liquid-liquid extraction method, the chromatographic separation was conducted on a trifunctional modified hydrophilic column. The mobile phases in the gradient program were 2.5% formic acid aqueous solution and acetonitrile. The multiple reaction monitoring (MRM) models and the isotope-labeled internal standards were used in the acquisition process. Good linearities and satisfying recovery rates were obtained in two sample matrices with good RSDs. The detection limits of GLY and AMPA were <2 μg L-1, which were close to those obtained in our previous research. The established method was applied to biological samples from five patients with glyphosate intoxication. The analysis of the trend for the concentration of GLY and AMPA in two biological samples was investigated, and the difference in the downward trend of AMPA in urine was found in patients with a relatively higher concentration of GLY in serum.