Abstract: With the rapid development of society and the gradual improvement of living standards, the problem of pesticide residue in food safety detection had aroused widespread concern. Among them, organophosphorus pesticide residues in food such as vegetables and fruits frequently occurred. Among them, methyl chlorpyrifos is one of the organophosphorus pesticides commonly used in agricultural production, which is causing widespread concern in pesticide residue detection. The purpose of this paper is to synthesize a stable isotope labelled methyl chlorpyrifos-D₆ as an internal standard reagent for accurate detection of pesticide residues. Using trichlorosulfate as the starting material and the cheap and easily available methanol-D₄ as a stable isotope labelling source, an important isotopically labelled dimethyl chloroth-iophosphate-D₆ was synthesized by a two-step nucleophilic substitution reaction as an intermediate. The isotopically labelled dimethyl chloroth-iophosphate-D₆ and 3,5,6-trichloropyridin-2-ol were used in the two-phase composite system to synthesize the stable isotope-labeled methyl chlorpyrifos-D₆ by phase transfer catalyst. The methyl chlorpyrifos-D₆ could be used as an internal standard reagent for pesticide residue detection. A better synthetic route was obtained by optimizing the experimental conditions such as the molar ratio of the reaction raw materials and the solvent. This synthetic route was safe, reliable, easy to operate, and had no special requirements for equipment, which was suitable for the laboratory scale. The product was identified by ¹H NMR, GC and GC-MS. The yield of methyl chlorpyrifos-D₆ with a purity of 98.3% and an isotope abundance of 99.0atom%D was 44.2% based on the consumed methanol-D₄.