Regardless of the emergent use of fluorescence as an analytical method to investigate biodiesel no detailed discussion about the origin of the visible fluorescence in different biodiesel samples has been reported. Here, UV-Vis absorption and fluorescence analysis of biodiesels (soybean, corn, canola, and sunflower), standard methyl esters (stearate, palmitate, oleate, linoleate and linolenate), phenolic antioxidants, beta-carotene, alpha-tocopherol, and chlorophyll were performed in order to investigate the molecules responsible for the fluorescence in biodiesel. Additionally, mid-infrared absorption, gas chromatography as well as C-13 and H-1 nuclear magnetic resonance measurements were also carried out. The findings reveal the presence of fluorescent molecules such as conjugated tetraenes in both the standard methyl esters and biodiesel, and so these minority compounds could explain the visible fluorescence of these biodiesel samples in the 350-500 nm range under UV excitation, excluding the contribution of free fatty acids, tocopherols, carotenoids, phenolic antioxidants and chlorophyll. The results also indicate that as higher the methyl linolenate content in the biodiesel as higher the concentration of conjugated tetraenes in the biodiesel sample, explaining the intense visible fluorescence observed mainly in the soybean and canola biodiesel. (C) 2013 Elsevier Ltd. All rights reserved.