Abstract: The uncertainty of standard solution could be influenced by many factors such as the preparation and dilution and so on, and thus affect the accuracy of pesticide residue detection. The paper took the practical application of 14 kinds of organophosphorus pesticides mixed standard solution as samples, used the calculation method of preparing the mixed standard solution process unit operational uncertainties (top down) to analyze all factors in the mixed standard solution preparation process. Evaluation of various components of the uncertainty was carried out and final expanded uncertainty was obtained. The results showed that preparing the stock solution into mixed standard solution by the precise pipette could get relatively small expanded uncertainty.