The invention discloses stable isotope-labeled chlorpyrifos and a derivative and synthetic preparation method thereof. An organic synthesis method is utilized, thiophosphoryl chloride is adopted as araw material, a reaction is performed on a stable isotope-labeled 3,5,6-trichloropyridine-2-ol sodium salt, and then a reaction is performed on the reaction product and stable isotope-labeled short-chain alcohol so as to obtain the stable isotope-labeled chlorpyrifos and the derivative of the stable isotope-labeled chlorpyrifos. After simple separation and purification are performed on the prepared stable isotope-labeled chlorpyrifos and the derivative, the chemical purity can reach 99% or above, and the isotope abundance can reach 99% or above; and the stable isotope-labeled chlorpyrifos andthe derivative can be applied to biological metabolism, food safety, environmental monitoring and other fields, and compared with foreign products, the stable isotope-labeled chlorpyrifos and the derivative have the advantages of good quality and low cost.