In order to evaluate the uncertainty of determination of Aflatoxin B₁ in peanut butter by high performance liquid chromatography. According to the uncertainty assessment described in CNAS—GL06-2006 Guidance on evaluation the uncertainty in chemical analysis and JJF 1059.1—2012 Evaluation and expression of uncertainty in measurement, the components of uncertainty in the measuring process were studied and analyzed, and the related expanded uncertainty was calculated and evaluated. The results of method showed the content of Aflatoxin B₁ in peanut butter was 25.12 μg/kg, the related expanded uncertainty was 2.1%, and the determination result was X=(25.12±0.528) μg/kg, k=2. Among the 6 factors of uncertainty, the content of targeted ions from liners fitting and sample preparation was the main factors which influenced the results mostly. Through reducing the amounts of these factors, the quality of the determination results can be increased. It could improve the accuracy of measurements in routine tests.