Abstract: Sudan Ⅱ is one of synthetic organic azo-compounds, which is abundantly used in industry for coloring. A variety of foodstuffs contaminated with Sudan dyes have been detected throughout Europe and Asia. This issue has become one focal point for addressing food safety. Sudan Ⅱ could produce aniline and its derivatives, which could attack the hepatocytes and lead to the toxic hepatic disease. For this reason, accurate and reliable determination of Sudan Ⅱ in foodstuff is required for the assurance of consumer’s healthiness. But the traceability of Sudan Ⅱ has not been settled yet. Qualitative and quantitative analysis of the major organic impurities in pure Sudan Ⅱ was performed in this work. The related organic impurities were qualitatively determined by high performance liquid chromatography-ion trap-time of flight mass spectrometry (HPLC-IT-TOF MS) and high performance liquid chromatography-triple quadrupole tandem mass spectrometry (HPLC-MS/MS). Full scan of LC/MS in positive ion mode was firstly executed to obtain the m/z values of the molecules. Then LC-MS/MS was carried out on target compounds to obtain as much structural information as possible. According to the first and the second mass spectra, retention time of standard materials and published reports, the impurities in Sudan Ⅱ were identified and their fragmentation pathways were concluded. Four unknown related organic impurities were observed in Sudan Ⅱ. From analysis of possible structures and fragmentation pathways of fragments, impurities 1 and 2 were successfully identified as SudanⅠ and Solvent Orange 2, respectively. The results were verified by standards. The possible molecular structures of impurity 3 and 4 were derived as isomers of Sudan Ⅱ. High performance liquid chromatography external standard method was used to determine the contents of the identified organic impurities. The results showed that there are 0.017% Sudan Ⅰ with the RSD of 1.6% and 0.258% Solvent Orange 2 with the RSD of 2.5% in pure Sudan Ⅱ substance. Impurities 3 and 4 were quantified in accordance with the Sudan Ⅱ response factor under DAD detector. The contents of impurities 3 and 4 are 0.012% and 0.028% with the RSD of 7.0% and 4.9%, respectively. Moreover, this method has instructive significance to the qualitative and quantitative analysis of pure Sudan dyes and other pure organic substances. Also it will assure the accuracy of determination of Sudan Ⅱ and lay the foundation for development of a national first grade certified reference material of Sudan Ⅱ.