The uncertainty of the determination of VA, V_D3 and VE content in infant nutritional rice flour by HPLC was evaluated. According to JF1059.1—2012 ＂evaluation and Representation of Measurement Uncertainty＂, a mathematical model for simultaneous determination of VA, V_D3 and VE content uncertainty in infant nutrition rice flour by HPLC was established, and the sources of uncertainty in the measurement process were analyzed, quantified and synthesized layer by layer. When the sampling volume was 5.000 g, the expanded uncertainty of VA, V_D3 and VE in the sample was 0.188 4, 0.009 3, 0.006 7 μg/g, and the measured content was(9.250 0±0.188 4), (0.114 0±0.009 3), (0.181 5±0.006 7) μg/g, respectively. The uncertainty of the final result was mainly caused by the preparation of standard solution reserve, fitting of standard working curve and recovery rate.