Abstract: The ¹³C-labeled synthesis of cholesterol-3,4-¹³C₂ was based on phenyl acetate-1,2-¹³C₂ and 4-cholesten-3-one. 4-Cholesten-3-one was reacted in a certain proportion solvent mixed by isopropanol and water to obtain the carboxylic acid intermediate through ring opening oxidation reaction using potassium permanganate and sodium periodate as oxidant, and then reacted with acetate under heat and reflux in nitrogen protection to produce the ring-closure carbonyl intermediate. The acetylation labeled intermediate was synthesized with the ring-closure carbonyl intermediate and phenyl acetate-1,2-¹³C₂ in tetrahydrofuran as solvent, using sodium hydride as catalyzer through acetylation reaction. Finally the cholesterol-3,4-¹³C₂ was obtained sequentially by carbon ring reconstruction reaction, esterification and reduction reaction with the acetylation labeled intermediate as raw material. The reduction reaction showed a certain degree of asymmetric synthesis phenomenon. The yield based on the amount of substance of acetate-1,2-¹³C₂ was 16.2%. The structure of target product was confirmed by MS, NMR and HPLC. Its chemical purity (HPLC) was higher than 96%, and isotopic abundance was higher than 98% (atom ¹³C). This product can be used as the stable isotope internal standard reagent for detection in the field of clinical mass spectrometry and biomedical. It can also be used as a raw material of the test kit for clinical mass spectrometry disease detection.