In the context of new alternative crop development in Europe, flavonoids were investigated in saw-wort, Serratula tinctoria L., a perennial Asteraceae which was used as a yellow dye until the 19th century. The phytochemical study described in this report indicates that leaves rather than stems should be used, and harvested at the end of the plant growing cycle, when flavonoids are particularly concentrated. Microspectrofluorometry showed a specific distribution of the flavonoid aglycone, luteolin in stomatal cells whereas the corresponding glycoside (luteolin-7-O-glucoside) was observed in palisade parenchyma cells. The flavonoids luteolin-4′-O-glucoside and 3-methylquercetin were isolated for the first time in S. tinctoria leaves and identified by NMR spectroscopy. The role of these flavonoids is discussed in this paper. Using a rapid and simple method, i.e. flavonoid histolocalization associated with UV, it was demonstrated that saw-wort contains high concentrations of luteolin derivatives and could be considered for use again as a natural dye.