To evaluate the uncertainty of the determination of vitamin C in fruits and vegetables by high performance liquid chromatography, a mathematical model was established to evaluate each uncertainty component, and the standard uncertainty and extended uncertainty of the synthesis were calculated. The results showed that the amounts of vitamin C in samples was 33.7 mg/100 g, and the expanded uncertainty was 1.4 mg/100 g (k=2). The uncertainties of the determination mainly come from the preparation of standard solution and the repeatability of determination.