Abstract: Hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferase (HQT), is the key enzyme catalyzing the final step in biosynthesis of caffeoylquinic acids. In order to promote the accumulation of tobacco polyphenols, we constructed the vetor P35S::SlHQT which was heterogeneously expressed in Nicotiana tabacum var. Samsun (SS). High performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS/MS) analysis were used to detect the content of polyphenols in tobacco leaves. The results showed that the concentrations of caffeoylquinic acids were increased by 14.98-fold; rutin and kaempferol rutinoside were increased by 11.47- and 9.32-fold respectively. And the transgenic tobacco exhibited no visible differences compared with wild-type tobacco. There were 4 types of CQAs in the transgenic lines:1-CQA, 3-CQA, 4-CQA, and 5-CQA (CGA) while only 3 types of CQAs were detected in the wild type tobacco:3-CQA, 4-CQA, and 5-CQA (CGA). These results suggested that SlHQT promoted the biosynthesis of 1-CQA and positively regulated the synthesis of flavonols as well as caffeoylquinic acids in tobacco.