Abstract: The reference material of caffeine in methanol-water for LC-MS calibration was developed, and uncertainty was evaluated. Using pure caffeine, chromatographic grade methanol, and laboratory first-grade water as raw materials, the purity of the pure caffeine product was verified by the mass balance method, and the standard method weight-volume method was used to prepare the reference material of the caffeine solution in methanol with a concentration of 9.94 mg/L. Liquid chromatography was used to compare the value of the 9.94 mg/L standard solutions of caffeine in methanol with the national certified reference material GBW(E)130453, and the uniformity, short-term stability, and long-term stability of the solution were studied. The results show that the developed 9.94 mg/L reference material of caffeine in methanol-water has good uniformity and stability, with a relative extended uncertainty of 2% (k=2) and a validity period of 12 months.