Objective To determine the chlorpyrifos in human blood by liquid chromatography-tandemmass spectrometry and to validate its application in poisoning cases. Methods The samples were extracted by a simple one-step protein precipitation procedure. Chromatography was performed on a Capcell Pack C18 mG II column (250 mm×2.0 mm, 5μm) using an isocratic elution of solvent A (0.1% formic acid-water with 2 mmol/L ammoniumacetate) and solvent B (methanol with 2 mmol/L ammoniumacetate) at 5∶95 (V∶V).Results The linearranged from5 to 500ng/mL (r=0.9987).Thelimitofdetection (LOD) and the lower limit of quantification (LLOQ ) were 2 ng/mL and 4 ng/mL , respectively. For this method, the precision and accuracy of intra-day and inter-day were <10% and 97.44%-101.10%, respectively. The re-sults in stability test of long-termfrozen were satisfied. The matrix effect, recovery and process efficien-cy were 64.97%-86.81%, 76.70%-85.52%, and 55.57%-66.58%, respectively. Conclusion This method can provide a rapid approach to chlorpyrifos extraction and determination in toxicological analysis of forensic and clinical treatment.