This study aimed to conduct measurement uncertainty assessment of a new method for determination of Sudan colorants(Sudan Ⅰ, Ⅱ, Ⅲ and Ⅳ) in food by high performance liquid chromatography(HPLC). Samples were extracted with organic solvents(hexane, 20% acetone) and first purified by magnesium trisilicate(2Mg O·3Si O2). The Sudan colorants(Sudan Ⅰ–Ⅳ) were also initially separated on C8 by gradient elution using acetonitrile and 0.1%(v/v) formic acid aqueous solution as the mobile phases and detected with diode-array detector(DAD). The uncertainty of mathematical model of Sudan Ⅰ, Ⅱ, Ⅲ, Ⅳ is based on EURACHEM guidelines. The sources and components of uncertainty were calculated. The experiment gave a good linear relationship over the concentration from 0.4 to 4.0 μg/m L and spiked recoveries were from 74.0% to 97.5%. The limits of determination(LOD) were 48, 61, 36, 58 μg/kg for the four analytes, respectively. The total uncertainty of Sudan colorants(Sudan Ⅰ, Ⅱ, Ⅲ and Ⅳ) was 810±30.8, 790±28.4, 750±27.0, 730±50.0 μg/kg, respectively. The recovery uncertainty was the most significant factor contributing to the total uncertainty. The developed method is simple, rapid, and highly sensitive. It can be used for the determination of trace Sudan dyes in food samples. The sources of uncertainty have been identified and uncertainty components have been simplified and considered. This study aimed to conduct measurement uncertainty assessment of a new method for determination of Sudan colorants (Sudan I, II, III and IV) in food by high performance liquid chromatography (HPLC). Samples were extracted with organic solvents (hexane, 20% ) and first purified by magnesium trisilicate (2Mg O.3Si O2). The Sudan colorants (Sudan I-IV) were also initially separated on C8 by gradient elution using acetonitrile and 0.1% (v / v) formic acid aqueous solution as the mobile The uncertainty of mathematical model of Sudan Ⅰ, Ⅱ, Ⅲ, Ⅳ is based on EURACHEM guidelines. The sources and components of uncertainty were calculated. The experiment gave a good linear relationship over the concentration from 0.4 to 4.0 μg / mL and spiked recoveries were from 74.0% to 97.5%. The limits of determination (LOD) were 48, 61, 36, 58 μg / kg for the four analytes, respectively. The total uncertainty of Sudan colorants (Sudan I, , Respectively. The recovery uncertainty was the most significant factor contributing to the total uncertainty. The developed method is simple, rapid, and highly sensitive. It can be used for the determination of trace Sudan dyes in food samples. The sources of uncertainty have been identified and uncertainty components have been simplified and considered.