Adulteration of extra virgin olive oil with sunflower oil is a major issue for the olive oil industry. In this paper, the potential of total synchronous fluorescence (TSyF) spectra to differentiate virgin olive oil from sunflower oil and synchronous fluorescence (SyF) spectra combined with multivariate analysis to assess the adulteration of virgin olive oil are demonstrated. TSyF spectra were acquired by varying the excitation wavelength in the region 270-720 nm and the wavelength interval (Deltalambda) in the region from 20 to 120 nm. TSyF contour plots for sunflower, in contrast to virgin olive oil, show a fluorescence region in the excitation wavelength range 325-385 nm. Fifteen different virgin olive oil samples were adulterated with sunflower oil at varying levels (0.5-95%) resulting in one hundred and thirty six mixtures. The partial least-squares regression model was used for quantification of the adulteration using wavelength intervals of 20 and 80 nm. This technique is useful for detection of sunflower oil in virgin olive oil at levels down to 3.4% (w/v) in just two and a half minutes using an 80-nm wavelength interval.